Tara M Stanne

Manish Kushwaha

Matthew Wand

Jesse E Taylor

Gloria Rudenko
email: gloria.rudenko@imperial.ac.uk

TbISWI regulates multiple Pol I transcribed loci and is present at Pol II transcription boundaries 2 in Trypanosoma brucei 3 4 5

Keywords: VSG expression sites, ISWI, African trypanosome, Strand Switch Regions (SSR), RNA polymerase I, chromatin remodeling, transcription control

The unicellular eukaryote Trypanosoma brucei is unusual in having very little transcriptional control. The bulk of the T. brucei genome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA polymerase I (Pol I) transcription units such as the VSG expression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/ SNF2 related chromatin remodelling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream and procyclic form T. brucei. Here, we show that TbISWI binds both active and silent ESs, but is depleted from the ES promoters themselves. TbISWI knock-down results in an increase in VSG transcripts from the silent VSG ESs. In addition to its role in repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribed VSG Basic Copy arrays and mini-chromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination, and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci, and which is particularly abundant at many Pol II transcription boundaries in T. brucei.

Introduction

As an early branching eukaryote Trypanosoma brucei, the causative agent of African Sleeping Sickness, has some unorthodox features. Most of the T. brucei genome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic arrays, and there is very little transcriptional control [START_REF] Clayton | Life without transcriptional control? From fly to man and back again[END_REF]. Another unusual feature is that RNA polymerase I (Pol I) transcribes not only the multi-copy ribosomal DNA (rDNA), but also the genes encoding the trypanosome major surface proteins Variant Surface Glycoprotein (VSG) and procyclin, which are controlled in a life-cycle specific fashion [START_REF] Günzl | RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei[END_REF][START_REF] Kooter | Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes[END_REF][START_REF] Rudenko | Alphaamanitin resistant transcription of protein coding genes in insect and bloodstream form Trypanosoma brucei[END_REF]. Bloodstream form T. brucei evades immune attack by the mammalian host through antigenic variation of its VSG surface coat by switching between different VSG variants. It is therefore critical for bloodstream form trypanosomes to tightly control expression of their VSG repertoire. Only one of about 1500 different VSG genes and pseudogenes is expressed in a mono-allelic fashion from one of about 15 telomeric VSG expression site (ES) transcription units [START_REF] Borst | Antigenic variation and allelic exclusion[END_REF][START_REF] Chaves | Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei[END_REF][START_REF] Pays | Antigenic variation in Trypanosoma brucei: facts, challenges and mysteries[END_REF]. Upon differentiation to the procyclic form insect mid-gut stage, the VSG coat is replaced by an invariant coat of procyclin [START_REF] Roditi | Dressed for success: the surface coats of insect-borne protozoan parasites[END_REF].

Very few regions of the trypanosome genome are non-transcribed. These include the silent VSG genes, which are located in sub-telomeric VSG Basic Copy arrays as well as at the telomeres of large, intermediate and mini-chromosomes [START_REF] Berriman | The genome of the African trypanosome Trypanosoma brucei[END_REF][START_REF] Melville | The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers[END_REF][START_REF] Wickstead | The small chromosomes of Trypanosoma brucei involved in antigenic variation are constructed around repetitive palindromes[END_REF]. Mini-chromosomes are primarily composed of arrays of 177 bp repeats and are transcriptionally silent [START_REF] Wickstead | The small chromosomes of Trypanosoma brucei involved in antigenic variation are constructed around repetitive palindromes[END_REF]. In addition, the rDNA spacer is an 8 kb non-transcribed region in between tandem arrays of rDNA transcription units [START_REF] White | Three small RNAs within the 10 kb trypanosome rRNA transcription unit are analogous to domain VII of other eukaryotic 28S rRNAs[END_REF]. Finally, the transcription units containing the T. brucei coat protein genes are turned off in the life-cycle stage where they are not needed. All VSG ESs are downregulated in procyclic form T. brucei where VSG is not expressed, and the procyclin on August 22, 2018 transcription units are silenced in bloodstream form T. brucei where it would be deleterious for an invariant surface protein to be expressed on the cell surface [START_REF] Vassella | Multiple procyclin isoforms are expressed differentially during the development of insect forms of Trypanosoma brucei[END_REF].

It is unclear how T. brucei keeps these transcriptionally inactive regions of the genome silent, as well as the extent to which epigenetic modifications such as chromatin remodeling are involved in this process. In the case of the ESs, it has recently been shown that the active ES in bloodstream form T. brucei is depleted of nucleosomes [START_REF] Figueiredo | Nucleosomes are depleted at the VSG expression site transcribed by RNA polymerase I in African trypanosomes[END_REF][START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF], indicating that chromatin remodeling plays a key role in ES activation. This is supported by the discovery that a number of proteins involved in epigenetic control play a role in ES regulation including the T. brucei ISWI homologue TbISWI [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF], the telomere binding protein RAP1 [START_REF] Yang | RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei[END_REF] and the histone methyltransferase DOT1B [START_REF] Figueiredo | A histone methyltransferase modulates antigenic variation in African trypanosomes[END_REF].

It has recently become clear that epigenetic marks are also present at the strand switch regions (SSRs) located between the polycistronic T. brucei Pol II transcription units [START_REF] Siegel | Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei[END_REF][START_REF] Wright | Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei[END_REF].

The divergent SSRs, which contain putative transcription start sites (TSS), are enriched in the histone variants H2AZ and H2BV as well as histones H3K4me3 and H4K10ac. Convergent SSRs containing putative transcription termination sites (TTS) are enriched in the histone variants H3v and H4v [START_REF] Siegel | Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei[END_REF]. The proposed histone labelling of a transcription stop site is another unique feature of the trypanosome genome.

In this study, we investigated the role of TbISWI in regulating gene expression in different genomic regions of T. brucei. We show that TbISWI binds in the T. brucei genome within silent DNA repeats, in silent and active transcription units transcribed by Pol I, as well as at Pol II-transcribed loci. We also find that TbISWI is particularly enriched at a number of SSRs

Materials and Methods

Trypanosomes.

Bloodstream and procyclic form T. brucei 427 cell lines were cultured according to [START_REF] Sheader | Bloodstream form-specific upregulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage[END_REF], a similar fashion, except that the parental cell line was T. brucei 29-13 (62). Reporter constructs containing eGFP were first transfected into 29-13 cell lines, followed by the TbISWI RNAi construct [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF].

except
TbISWI was epitope tagged at the C-terminus with a triple myc epitope using the pMoTAG43M vector [START_REF] Oberholzer | A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes[END_REF]. Epitope tagging was performed in bloodstream form T. brucei 221GPI(VO2+) cells [START_REF] Sheader | Bloodstream form-specific upregulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage[END_REF] and in wild type procyclic T. brucei. In order to ensure functionality of the tagged TbISWI, cell lines were also made where the second allele of TbISWI was knocked out using the pSpot5KOPhleo vector.

Constructs.

The sequences of all primers used for cloning are shown in Supplementary Table 1. The fragments used to target the reporter constructs to different loci are: (i) a mini-chromosomal 177 bp repeat sequence from p2T7 Ti -177 ( 59), (ii) a VSG118 Basic Copy array fragment amplified by PCR from T. brucei genomic DNA (gDNA) using primers BCVSG118-863s and BCVSG118-2025as, (iii) an rDNA spacer targeting fragment isolated from p2T7 Ti A/GFP [START_REF] Lacount | Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis[END_REF], and (iv) a procyclin targeting fragment amplified by PCR from T. brucei gDNA using primers EP1_UP_2480s and EP1_UP_3991as. Constructs targeting the first two loci were made with (+ESp) and without (-ESp) an ES core promoter isolated from rDES1 [START_REF] Rudenko | VSG gene expression site control in insect form Trypanosoma brucei[END_REF]. The reporter gene cassette consists of an eGFP ORF, with a tubulin splice signal at its 5'-end and a tubulin polyadenylation signal at its 3'-end. The drug resistance cassette consists of a drug resistance gene flanked by a 5'-tubulin splice signal and a 3'-actin polyadenylation signal and is driven by an rDNA promoter [START_REF] Sheader | Delineation of the regulated Variant Surface Glycoprotein gene expression site domain of Trypanosoma brucei[END_REF]. The reporter constructs all contained the blasticidin resistance (BlastR) gene as the drug marker, however the rDNA spacer targeting constructs were also made with the blot mapping (Sup. Fig. 1C). Reporter construct integration into the VSG118 Basic Copy array, the procyclin locus and the tubulin array was verified by PCR (Sup. Fig. 3C, Sup. Fig. 5B and Sup. Fig. 8B) (primers listed in Sup. Table 2). Integration into the rDNA spacer was confirmed by Southern blot analysis (Sup. Fig. 7B)(Sup. Table 3).

For chromatin immunoprecipitation (ChIP) studies, one of the endogenous TbISWI alleles was tagged at the C-terminal end with a triple myc epitope as follows: The C-terminal end of TbISWI, excluding the stop codon, was amplified from T. brucei 427 genomic DNA using Spot5CTtag_s and Spot5tag4934_as to yield a 701 bp fragment (position 2780-3480 of the TbISWI open reading frame). For the downstream region, an 827 bp fragment was produced using primers Spot5DRtag_s and Spot5DRtag_as. Both of these fragments were cloned into the pMoTAG43M vector, containing HygR for selection [START_REF] Oberholzer | A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes[END_REF].

The untagged allele of TbISWI was knocked-out through replacement with PhleoR to ensure functionality of myc-tagged TbISWI. A 499 bp fragment immediately upstream of the TbISWI open reading frame was amplified from T. brucei 427 genomic DNA using Spot5-957s-UPF and Spot5-1455as-US. For the downstream region, a 586 bp fragment was amplified using primers Spot5-4935s-DSF and Spot5-5520as-DS. These fragments were cloned on either side of PhleoR in the pBlueScript vector to produce pSpot5KOPhleo. The MC 177 TbISWI-A RNAi construct used for all reporter lines is described in [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF], and the MC 177 Tb11.01.2500 RNAi construct is described in the Supplementary Material. Protein lysates were made from bloodstream and procyclic form T. brucei essentially as described [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF] an anti-histone H3 antibody (ab1791; AbCam) as a positive control, no antibody as a negative control, and a monoclonal anti-myc antibody (clone 9E10, M5546; Sigma) to detect TbISWI.

The protein-DNA complexes were incubated with protein A beads (CL4B beads, GE Healthcare) and eluted in 1% SDS/0.1M NaHCO 3 . DNA was purified using QIAquick PCR purification kit (QIAGEN). Quantitative PCR (qPCR) was used to amplify regions of interest using the LightCycler ® 480 Real-Time PCR System (Roche) (primer details in Sup. Tables 4 and5). ChIP material was also analyzed on slot blots using radiolabeled probes as described previously [START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF]. Hybond-XL membranes (GE Healthcare) were hybridized with probes specific for the 177 bp repeats (61), the 50 bp repeat regions flanking VSG ESs [START_REF] Zomerdijk | The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei[END_REF] or the telomere repeats [START_REF] Blackburn | Identification of a telomeric DNA sequence in Trypanosoma brucei[END_REF]. Blots were quantitated using a PhosphorImager (BioRad) and the percentage of DNA immunoprecipitated was determined using QuantityOne software (BioRad). Signals are expressed as a percentage of input as described above.

RNA isolation and quantitative reverse-transcriptase PCR.

The bloodstream form T. brucei 121-SA1 cell line ( 21 at various time points using the RNeasy kit (Qiagen). RNA was DNase-treated using the TURBO DNA-free kit (Ambion) and cDNA was synthesized using the Omniscript RT kit (Qiagen) with random hexamer primers (Promega). qPCR was performed on a LightCycler ® 480

Real-Time PCR System using LightCycler ® 480 SYBR Green I Master Mix (Roche) in triplicate. The reaction conditions for each primer pair were individually optimized to obtain a single product of the correct size. The sequences of the primers used are detailed in Sup. Table 4. Control reactions without reverse transcriptase (RT) were made using DNAse-treated RNA from each time point. Levels of transcript were plotted as fold increase relative to the 0 hour time point after subtraction of the no RT controls. Three independent experiments were performed, and the standard deviation is indicated with error bars.

Results

TbISWI binds silent DNA repeats as well as the VSG Basic Copy arrays.

TbISWI was originally identified as binding the 177 bp repeats in DNA affinity purification experiments, and was subsequently shown to be involved in silencing VSG ESs in both bloodstream and procyclic form T. brucei [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF]. As the 177 bp repeats are primarily located in the transcriptionally silent mini-chromosomes (61), we investigated whether TbISWI binds to and represses other silent areas of the T. brucei genome. We performed chromatin immunoprecipitation (ChIP) analysis using an epitope-tagged copy of TbISWI. Bloodstream and procyclic form T. brucei lines expressing a TbISWI-myc fusion protein from one endogenous TbISWI allele were generated. Expression of the myc-tagged TbISWI was verified using Western blot analysis (Fig. 1A). Functionality of the myc-tagged TbISWI was confirmed by ChIP analysis was performed on bloodstream form cell lines containing either wild type or myc-tagged TbISWI. An Į-myc antibody was used to immunoprecipitate TbISWI, an Įhistone H3 antibody was used as a positive control, and no antibody was used as a negative control. Input chromatin and immunoprecipitated material were analyzed using either slot blots probed for repetitive regions, or by quantitative PCR (qPCR) for individual genes. As expected, we found that TbISWI binds the 177 bp repeats (Fig. 1B) with 0.05% of the input immunoprecipitated (Fig. 1C). TbISWI was also found to bind other silent regions including the 50 bp repeat arrays located upstream of VSG ESs (about 0.04% immunoprecipitated) [START_REF] Sheader | Delineation of the regulated Variant Surface Glycoprotein gene expression site domain of Trypanosoma brucei[END_REF][START_REF] Zomerdijk | The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei[END_REF], the telomere repeats (0.01% immunoprecipitated) and the subtelomeric VSG118 Basic Copy array (0.04% immunoprecipitated). Significant amounts of TbISWI were therefore found binding all of these nontranscribed regions (P< 0.05; see Supplementary Table 6 for statistical analyses). Similar results were obtained using procyclic form cells (data not shown). The minichromosomes and the VSG Basic Copy arrays house the silent VSG repertoire used during antigenic variation. As TbISWI was found binding both of these regions at similar levels, we next investigated the role of TbISWI in silencing these areas of the genome. Basic Copy Array, the EP procyclin locus, the rDNA spacer or the tubulin transcription unit (Fig. 2). The advantage of using eGFP to monitor for transcription is that gene expression at the population level can be analysed using flow cytometry. This allows one to uncover cell-cycle specific effects, which is not straightforward with a reporter like luciferase.

TbISWI is involved in down

Constructs integrated into the mini-chromosomes or the VSG118 Basic Copy array, either did (+) or did not (-) contain an ES promoter (ESp). Unlike other T. brucei Pol I promoters, the ES promoters are unusually small and lack an upstream control element [START_REF] Pham | A detailed mutational analysis of the VSG gene expression site promoter[END_REF], rendering transcription highly sensitive to the chromatin state of their genomic context [START_REF] Horn | Position-dependent and promoter-specific regulation of gene expression in Trypanosoma brucei[END_REF][START_REF] Urmenyi | PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei[END_REF]. We therefore used an ES promoter as a "probe" for repressed chromatin. These T. brucei reporter cell lines were subsequently transfected with a TbISWI RNAi construct allowing tetracycline inducible knock-down of TbISWI. See Materials and Methods for details on the various constructs and cell lines.

To determine whether TbISWI has a role in silencing the T. brucei mini-chromosomes, we integrated an eGFP reporter gene into the 177 bp repeat arrays in bloodstream form T. brucei (Fig. 2Ba, BF-MCe in Fig. 3A, Sup. Fig. 1A), and made a similar reporter line with an ES promoter upstream of eGFP (BF-MCESp in Fig. 3B, Sup. Fig. 1B). Correct integration of eGFP was confirmed by Pulsed Field Gel analysis and Southern blot mapping (Sup. Fig. 1C). Both of these reporter lines were transfected with a TbISWI RNAi construct [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF] and two independent RNAi clones per line were analysed (BF-MCe1, BF-MCe2 and BF-MCESp1, BF-MCESp2).

Western blot analysis confirmed TbISWI protein knock-down after the induction of TbISWI RNAi (Sup. 3D, Sup. Fig. 3). Similar to the cell lines where the reporter construct was integrated in mini-chromosomes, there was no significant increase in eGFP expression seen in the absence of an ES promoter (BF-VBe1 and BF-VBe2 in Fig. 3C).

However, if an ES promoter was present (BF-VBESp1 and BF-VBESp2 in Fig. 3D) approximately six-fold derepression of eGFP was observed after the induction of a block in TbISWI synthesis.

Comparable experiments were also performed in procyclic form T. brucei, and similar results were obtained (Sup.

TbISWI is present in both silent and active VSG ESs but is depleted from ES promoters.

The active VSG is transcribed by Pol I from one of about 15 highly similar polycistronic VSG ES transcription units (depicted schematically in Fig. 4A) [START_REF] Hertz-Fowler | Telomeric expression sites are highly conserved in Trypanosoma brucei[END_REF]. We determined the distribution of TbISWI along the length of these VSG ESs using ChIP on wild type or myctagged TbISWI cell lines followed by qPCR as described earlier. Using six different primer pairs (a-f in Fig. 4A and Fig. 4B), that detect most, if not all VSG ESs (i.e. the 14 silent ESs in addition to the one active ES), we determined that TbISWI is present at significant levels (P<0.05) along the length of the ES, with the exception of the "core" ES promoter [START_REF] Jefferies | Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level[END_REF] where TbISWI is relatively depleted (primer pair "a" in Fig. 4A and4B) (P<0.05; Sup. Table 7).

Primer pair "a" is located immediately upstream of the putative site of ES transcription initiation.

The relative depletion of TbISWI at ES promoters is particularly striking, considering that most if not all ESs can be detected with these particular primers. It is thought that the VSG ES "core" promoters of the 14 'silent' ESs contain 'paused' Pol I complexes which lead to a low amount of transcription (Fig. 4B) [START_REF] Ansorge | Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms[END_REF][START_REF] Rudenko | VSG gene expression site control in insect form Trypanosoma brucei[END_REF][START_REF] Vanhamme | Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei[END_REF]. It is possible that these 'paused' Pol I transcription complexes result in exclusion of TbISWI. This pattern of TbISWI binding is similar to that observed for histone H3 at this locus [START_REF] Figueiredo | Nucleosomes are depleted at the VSG expression site transcribed by RNA polymerase I in African trypanosomes[END_REF][START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF], and is consistent with TbISWI playing a role in nucleosome sliding and remodeling (30) [START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF].

Having determined the relative distribution of TbISWI along the VSG ESs (Fig. 4A Sup. Table 8) were found binding both active and silent ESs at approximately comparable levels (Fig. 4D).

We had shown earlier that TbISWI is important for silencing genes directly downstream of a silent ES promoter, although in those experiments we had not found clear evidence for an increase in transcripts from telomeric VSGs present in a number of silent ESs [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF]. Considering that TbISWI binds along the length of VSG ESs (Fig. 4), we wondered whether it could play a role in keeping the entire ES silent, including the telomeric VSG. We therefore decided to reinvestigate this question using newly available ES sequence data [START_REF] Hertz-Fowler | Telomeric expression sites are highly conserved in Trypanosoma brucei[END_REF].

Depleting TbISWI leads to an increase in previously silent telomeric VSG transcripts.

The repertoire of 15 VSG ESs from T. brucei 427 has recently been sequenced [START_REF] Hertz-Fowler | Telomeric expression sites are highly conserved in Trypanosoma brucei[END_REF]. This ). These results agree with previously published data [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF]. Transcript levels for Ȗ-tubulin remain relatively unchanged after TbISWI knock-down, which was also observed using a reporter construct integrated into the Į-ȕ tubulin array (Fig. 6E). Transcripts from the active VSG121 decrease by about 20%.

We next monitored transcripts from eleven silent ES-located VSGs as well as VSG118, which is located in a VSG Basic Copy array (Fig. 5B). Transcripts from all except for one of the telomeric ES-located VSGs increased significantly (P<0.05) after depletion of TbISWI for up to 72 hours. The observed increase in VSG transcripts varied between the different VSGs from fivefold (for VSG16) to 70-fold (for VSG1.8) (Sup. Table 9). Variability in the degree of derepression of different ES-located silent VSGs was also observed after the knock-down of RAP1, and presumably reflects a hierarchy of different VSG activation frequencies [START_REF] Yang | RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei[END_REF].

Interestingly, no statistically significant increase in VSG14 transcript was observed after TbISWI knock-down. VSG14 is located in a drastically truncated ES (BES8), which contains only ESAG7 [START_REF] Hertz-Fowler | Telomeric expression sites are highly conserved in Trypanosoma brucei[END_REF]. We have never found evidence for activation of BES8 in vitro [START_REF] Hertz-Fowler | Telomeric expression sites are highly conserved in Trypanosoma brucei[END_REF], and it is likely that this tiny ES is dysfunctional.

Transcript levels from the VSG118 Basic Copy also did not increase significantly after TbISWI knock-down, presumably as there are no promoters (which could be derepressed) in this area of the genome. In addition, there does not appear to be an increase in levels of fortuitous initiation of transcription in the VSG Basic Copy arrays. A comparable increase in telomeric ES located VSG transcripts was also observed using the T. brucei T3-SA1 cell line, where the

TbISWI binds within Pol I and Pol II transcription units but is only involved in silencing

Pol I transcribed loci.

Pol I not only transcribes VSG genes, but also the EP and GPEET procyclin genes encoding the invariant surface proteins of insect form T. brucei [START_REF] Günzl | RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei[END_REF][START_REF] Kooter | Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes[END_REF][START_REF] Roditi | Dressed for success: the surface coats of insect-borne protozoan parasites[END_REF][START_REF] Rudenko | Alphaamanitin resistant transcription of protein coding genes in insect and bloodstream form Trypanosoma brucei[END_REF], as well as the rDNA. In bloodstream form T. brucei the procyclin loci are downregulated, whereas in procyclic T. brucei they are actively transcribed [START_REF] Vassella | Multiple procyclin isoforms are expressed differentially during the development of insect forms of Trypanosoma brucei[END_REF]. As TbISWI was found to bind along both silent and active VSG ESs, we investigated TbISWI occupancy in one of the procyclin transcription units (depicted schematically in Fig. 6A) in both life cycle stages. Using ChIP-qPCR we found that significant amounts of TbISWI (P<0.05; Sup. Table 10) bind the EP procyclin coding region in both bloodstream and procyclic form T. brucei. TbISWI is relatively depleted from the EP promoter (Fig. 6A) in bloodstream form T. brucei (P<0.05), as was also observed at the promoters of the many 'silent' ESs which contain paused Pol I complexes (Fig. 4B) [START_REF] Ansorge | Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms[END_REF][START_REF] Rudenko | VSG gene expression site control in insect form Trypanosoma brucei[END_REF][START_REF] Vanhamme | Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei[END_REF]. It has been proposed that 'paused' Pol I transcription complexes are also present on 'silent' procyclin promoters in bloodstream form T. brucei [START_REF] Vanhamme | Stimuli of differentiation regulate RNA elongation in the transcription units for the major stage-specific antigens of Trypanosoma brucei[END_REF], possibly resulting in the displacement of TbISWI from this region.

We next investigated whether TbISWI is involved in silencing the procyclin locus in bloodstream form T. brucei. To address this, we created a reporter cell line with eGFP targeted downstream of an endogenous procyclin promoter (Fig. 6B, Sup. Fig. 5). This cell line was transfected with a TbISWI RNAi construct and two independent clones were isolated. TbISWI protein knock-down was confirmed by Western blot analysis (Sup. used to monitor levels of eGFP after TbISWI knock-down. After depleting TbISWI for four days there was a six-fold increase in eGFP expression (Fig. 6B). These results are similar to the levels of derepression observed in the mini-chromosomes and VSG Basic Copy array (Fig. 3B and 3D). A similar experiment was carried out in procyclic form T. brucei where depleting TbISWI led to a modest 2.5-fold derepression (Sup. Fig 6A). Therefore, TbISWI appears to play a role in modulating transcription of the EP procyclin locus in both life cycle stages of T. brucei.

In mammals, the ISWI containing nucleolar remodelling complex (NoRC) mediates silencing of the inactive rRNA genes [START_REF] Santoro | The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription[END_REF]. We were therefore interested whether TbISWI binds in and around T. brucei rDNA transcription units (Fig. 6C). ChIP was performed in bloodstream form T. brucei and qPCR was used to detect binding to four regions within the rDNA locus. In addition, two Pol II transcribed genes were analysed: the gene encoding the large subunit of Pol I and Į-tubulin. Significant levels of TbISWI were found binding all of these regions (P< 0.05; Sup. Table 11). TbISWI was particularly enriched in the transcriptionally inactive rDNA spacer (primer pair "a" in Fig. 6C) compared with the 18S rRNA coding region (primer pair "d", Fig. 6C) (P<0.05). Similar results were found in procyclic T. brucei (result not shown). TbISWI distribution in the rDNA closely resembled that of histone H3 (51), consistent with ISWIcontaining complexes playing a role in nucleosome remodelling.

To determine if TbISWI plays a role in silencing the rDNA locus, we created a bloodstream form reporter line containing a construct with eGFP downstream of an ES promoter located in an rDNA intergenic region between two rDNA transcription units (Fig. 6D, Sup. Fig. 7). After two days induction of TbISWI RNAi there was a four-fold increase in levels of eGFP (Fig. 6D). Comparable derepression was observed in procyclic T. brucei (Sup. Fig. 6B). These We see that in most cases in these different bloodstream form T. brucei reporter lines, T.

brucei TbISWI protein is greatly depleted 24 hours after induction of TbISWI RNAi (Sup. Fig. 2). An observable growth arrest is typically seen about 48 hours after induction of TbISWI RNAi (Sup. Fig. 10). The timing of this growth inhibition coincides with the timepoint of maximal derepression of eGFP in most of these reporter cell lines, with the exception of the minichromosome reporter line, where maximal derepression of eGFP was seen a bit later (after three days). This delay between the reduction of TbISWI protein to very low levels, and the appearance of a growth or derepression phenotype could indicate that even small amounts of TbISWI protein are sufficient to maintain normal levels of silencing [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF].

Most of the T. brucei genome is constitutively transcribed by Pol II as extensive polycistronic units. As TbISWI was found to bind to two Pol II transcribed genes (Fig. 6C), we investigated if TbISWI could play a role in regulating Pol II transcription. We inserted an eGFP reporter construct into the tubulin array, depleted the cells of TbISWI, and monitored levels of eGFP (Fig. 6E, Sup. Fig. 8). We found no significant change in the levels of eGFP after blocking TbISWI synthesis in both life cycle stages of T. brucei (Fig. 6E and Sup. Fig. 6C).

TbISWI is enriched in Pol II strand switch regions.

Recent data have identified enrichment of various modified histones and histone variants in regions between polycistronic Pol II transcription units in T. brucei [START_REF] Siegel | Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei[END_REF][START_REF] Wright | Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei[END_REF]. As ISWI complexes are involved in Pol II transcription initiation and termination in other organisms [START_REF] Mellor | ISWI complexes in Saccharomyces cerevisiae[END_REF], at strand switch regions (SSR). We analysed three 'divergent' and three 'convergent' SSRs on T. brucei chromosome 10 using ChIP-qPCR analysis (Fig. 7A). Regions chosen included those within the SSRs (primers c-e), in the genes directly flanking the SSRs (primers b and f), as well as in genes at the approximate midpoint of the flanking polycistronic units (primers a and g).

this
Using anti-histone H3 antibody, histone H3 was found to be relatively uniformly distributed over divergent and convergent regions (Sup. Fig. 9).

The SSRs between two diverging Pol II transcription units are likely to contain Pol II promoters. Significant levels of TbISWI (P<0.05) were found binding three 'divergent' SSRs (D1-D3) and flanking transcription units (Fig. 7B; Sup. Table 12). TbISWI was found to be significantly enriched in the 'divergent' SSRs (primers c-e) compared with genes within the neighbouring polycistronic transcription units (primers a and g) in divergent regions D1 and D2, although this was less clear for divergent region D3.

The SSRs between two converging Pol II transcription units are likely to contain Pol II terminators. Lower but significant amounts of TbISWI (P<0.05; Sup. Table 13) were found binding three 'convergent' SSRs (C1-C3) and flanking transcription units (Fig. 7C). There was evidence for enrichment of TbISWI on the SSRs of 'convergent' regions C2 and C3, though this was not found for region C1. TbISWI therefore appears to be enriched at many (particularly divergent) SSRs, specifically co-localizing with nucleosomes enriched in modified histones and histone variants known to have roles in regulating transcription by RNA Pol II in other organisms [START_REF] Barski | High-resolution profiling of histone methylations in the human genome[END_REF][START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF][START_REF] Siegel | Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei[END_REF]. TbISWI therefore appears to be associated with both Pol I and Pol II transcription units in T. brucei. Members of the ISWI family of chromatin remodelers are involved in a wide variety of processes ranging from transcription activation, repression and termination, as well as the maintenance of higher order chromatin structure and DNA replication [START_REF] Corona | Multiple roles for ISWI in transcription, chromosome organization and DNA replication[END_REF]. The specialized function of individual ISWI complexes depends on the protein partners associated with ISWI.

Discussion

Here, we find that TbISWI is involved in downregulating regions containing silent VSGs as well as other Pol I transcription units. TbISWI was found to be particularly enriched in many Pol II strand switch regions, which are the proposed sites of Pol II transcription initiation and termination and are known to contain histone variants and specific histone modifications [START_REF] Siegel | Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei[END_REF][START_REF] Wright | Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei[END_REF].

TbISWI is involved in preventing transcription of the silent VSG repertoire.

For antigenic variation to work, it is critical for bloodstream form T. brucei to tightly control expression of its extensive VSG repertoire. VSGs are located at most, if not all, telomeres, as well as in sub-telomeric VSG Basic Copy arrays [START_REF] Berriman | The genome of the African trypanosome Trypanosoma brucei[END_REF][START_REF] Melville | The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers[END_REF][START_REF] Wickstead | The small chromosomes of Trypanosoma brucei involved in antigenic variation are constructed around repetitive palindromes[END_REF]. We found that TbISWI binds mini-chromosomes, the VSG118 Basic Copy array, as well as VSG ESs. Using eGFP reporter constructs inserted into the 177 bp repeats of mini-chromosomes and the VSG118

Basic Copy array, we found that TbISWI depletion does not lead to a significant increase in transcription from these nontranscribed genomic regions. It is possible that even if TbISWI depletion results in a more "open" chromatin configuration, the absence of promoters in these regions prevents a more significant increase in transcription.

In order to study the role of TbISWI in maintaining a repressed chromatin state at these Pol I transcribes the multi-copy rDNA transcription units as well as the VSG and procyclin genes. Protein coding genes transcribed by Pol I are tightly regulated, unlike the constitutively transcribed Pol II transcription units [START_REF] Clayton | Life without transcriptional control? From fly to man and back again[END_REF]. Silent ESs in both bloodstream and procyclic form T. brucei have "poised RNA polymerases" at their promoters, and low levels of non-processive transcription [START_REF] Ansorge | Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms[END_REF][START_REF] Rudenko | VSG gene expression site control in insect form Trypanosoma brucei[END_REF][START_REF] Vanhamme | Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei[END_REF]. A similar situation is also seen in the procyclin loci that are transcribed at a low level in bloodstream form T. brucei [START_REF] Vanhamme | Stimuli of differentiation regulate RNA elongation in the transcription units for the major stage-specific antigens of Trypanosoma brucei[END_REF]. Using ChIP-qPCR we show that TbISWI is present on silent VSG ESs and on the silent EP1 procyclin locus in bloodstream form T. brucei, but is relatively depleted from the ES (both silent and active) promoters as well as procyclin promoters. One possibility is that poised RNA polymerase complexes on these different promoter regions displace TbISWI from binding. Alternatively, as TbISWI is a nucleosome remodeler, this relative absence could be a consequence of the relative depletion of nucleosomes observed on ES and procyclin promoters [START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF].

Both of these Pol I transcribed loci are derepressed when TbISWI synthesis is blocked.

While silent ESs are derepressed 30-60 fold in bloodstream form and 10-17 fold in procyclic form T. brucei [START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF], a silent procyclin locus in bloodstream form cells is derepressed about 6fold. Surprisingly, expression from even the active procyclin locus is upregulated by 2.5 fold in procyclic T. brucei after TbISWI depletion. This suggests that EP1 procyclin is not transcribed at its maximal capacity in procyclic form T. brucei due to a TbISWI mediated repressive Our ChIP-qPCR data also shows that TbISWI binds the active ES in bloodstream form T.

brucei as well as the active EP1 procyclin locus in procyclic form T. brucei. However, the fact that TbISWI is found binding both active and silent transcription units does not necessarily argue that it is doing the same thing in these two very different places, as ISWI complexes can have different functions depending on exactly which proteins ISWI is partnered up with. In Saccharomyces cerevisiae an ISWI complex is present on the active rDNA transcription units which facilitates transcription [START_REF] Jones | RNA polymerase I in yeast transcribes dynamic nucleosomal rDNA[END_REF]. In fact the ISWI protein Isw1p is a member of two different ISWI chromatin remodelling complexes: the Isw1a complex (consisting of Isw1p partnered with It has been suggested that chromatin remodelling is required to allow for the recognition of the histone N-terminal tails by histone modifying enzymes [START_REF] Krajewski | SET domains of histone methyltransferases recognize ISWI-remodeled nucleosomal species[END_REF]. Evidence is mounting that ISWI-containing complexes are necessary for histone methyltransferases and acetyl transferases to gain access to their histone targets [START_REF] Carre | The Drosophila NURF remodelling and the ATAC histone acetylase complexes functionally interact and are required for global chromosome organization[END_REF][START_REF] Krajewski | SET domains of histone methyltransferases recognize ISWI-remodeled nucleosomal species[END_REF][START_REF] Li | NoRC-dependent nucleosome positioning silences rRNA genes[END_REF]. Histone variant H2A.Z-containing nucleosomes are less stable and help maintain an accessible chromatin structure at transcription start sites [START_REF] Jin | Nucleosome stability mediated by histone variants H3.3 and H2A[END_REF]. ISWI remodelling complexes have also been found to be involved in histone variant H2A.Z replacement [START_REF] Hanai | RSF governs silent chromatin formation via histone H2Av replacement[END_REF][START_REF] Wan | Role of the histone variant H2A.Z/Htz1p in TBP recruitment, chromatin dynamics, and regulated expression of oleate-responsive genes[END_REF] and have greater activity on H2A.Z-containing nucleosomes

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 1140 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

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 115 Pol II transcription units containing the presumed Pol II transcription initiation and termination sites. Using eGFP reporter constructs inserted into various genomic loci, we on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-of 40 demonstrate that TbISWI is involved in repressing an ES promoter in regions containing silent VSGs, including the VSG Basic Copy arrays and the mini-chromosomes, as well as genes transcribed by Pol I including procyclin. Knock-down of TbISWI resulted in an increase in VSG transcripts from the silent telomeric VSG ESs, indicating that it plays a role in keeping the entire ES silent. Our results indicate that the chromatin remodeler TbISWI is involved in transcription by both Pol I and Pol II in T. brucei.

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 1140 that the bloodstream form was grown in the presence of 20% foetal calf serum (FCS) without the addition of SerumPlus. The bloodstream form T. brucei 121-SA1 cell has an active VSG121 ES, eGFP in the silent VSG221 ES, and a TbISWI RNAi construct (described in (21)). Additional bloodstream form T. brucei lines were made containing a reporter eGFP gene inserted into various loci including: the VSG Basic Copy array, the 177 bp repeats of minichromosomes, the procyclin locus, the rDNA spacer, and the tubulin array. All bloodstream form reporter lines were derived from T. brucei S16.221, a derivative of the T. brucei S16 'single marker' cell line (62). S16.221 was obtained by transfecting S16 with the pHNES221Pur1.6 construct (unpublished) that inserts a puromycin resistance gene downstream of the active VSG221 ES promoter. Next, the relevant eGFP containing reporter construct was transfected into S16.221, followed by the MC 177 TbISWI-A RNAi construct (21). The lineage of the different cell lines used for the reporter assays is shown in Fig. 2. TbISWI RNAi was induced by incubating the cells with 750 ng ml -1 tetracycline. All procyclic form reporter lines were made in on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

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 117 phleomycin resistance (PhleoR) gene. The tubulin array targeting reporter construct was derived on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-of 40 from pGad8-tubulin (60) by replacing the hygromycin resistance gene (HygroR) by BlastR. All sequences are available upon request. Verification of the reporter construct integration into the different genomic loci is described in the Supplementary Material. Integration of reporter constructs into mini-chromosomes was verified by Pulsed Field Gel (PFG) analysis and Southern

on August 22 , 11 Page 8 of 40

 221140 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-Western blotting.

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 1140 Flow cytometry was performed on T. brucei cell lines containing an eGFP reporter gene in different genomic locations after induction of RNAi against TbISWI for the times indicated. A Becton Dickinson FACS Calibur (BD Biosciences) was used to monitor the fluorescence of the cells in the FL-1 channel. The mean of 100,000 events was calculated using CellQuest software (BD Biosciences). Fold derepression was determined by dividing the mean FL-1 values of RNAi induced cultures at each time point by the mean FL-1 values of uninduced cultures. For each cell line, three independent experiments were performed and the standard deviations are indicated with error bars.ChIP assays and quantitative PCR.ChIP was performed in T. brucei cells expressing wild type or myc-tagged TbISWI essentially as described[START_REF] Stanne | Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes[END_REF]. In brief, cultures were fixed in 1% formaldehyde for 1 hour at room temperature and chromatin from 7 x 10 7 cell equivalents was incubated for[START_REF] Goldmark | The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p[END_REF] hours with on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

  For the quantitation of TbISWI ChIP experiments, signals are expressed as a percentage of input immunoprecipitated using an anti-myc antibody after subtraction of signal from the no antibody control for both the wild type TbISWI and myc-tagged TbISWI cell lines. Quantitation of histone H3 shows the percentage of input immunoprecipitated after subtraction of signal from the no antibody control. The data shown is from bloodstream form T. brucei and is the average of three to five independent ChIP experiments, with the standard deviation indicated with error bars. ChIP-qPCR was also carried out in procyclic form T. brucei and produced very similar results to those found for bloodstream form T. brucei in all regions analysed.

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 1140 ) was used for quantitative reversetranscriptase PCR (qRT-PCR). RNAi against TbISWI was induced, and total RNA was isolated on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

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 1140 knocking out the other TbISWI allele. The TbISWI-myc/ TbISWI_KO strains grew normally on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-to cells expressing wild type TbISWI in both life cycle stages (data not shown). The myc tag therefore did not detectably interfere with TbISWI function. This cell line was used for all successive ChIP experiments.

  -regulating transcription in T. brucei mini-chromosomes and VSG Basic Copy arrays. In order to investigate the role of TbISWI in silencing, we made T. brucei reporter cell lines where the parental bloodstream form T. brucei S16.221 line or the procyclic form T. brucei on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-11 Page 12 of 40 29-13 line had an eGFP containing construct inserted into the mini-chromosomes, the VSG118

Fig. 2 )

 2 , and flow cytometry was used to monitor eGFP expression. Four days after the induction of TbISWI RNAi there was no significant increase of eGFP expression observed in on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-11 Page 13 of 40 the lines not containing an ES promoter (BF-MCe1 and BF-MCe2 in Fig. 3A) in comparison to the negative control (BF-MCe). This indicates that blocking TbISWI synthesis does not lead to a significant increase in fortuitous initiation of transcription in the mini-chromosomes. In cell lines where an ES promoter was present upstream of eGFP, knock-down of TbISWI leads to more than six-fold derepression of eGFP (BF-MCESp1 and BFMCESp2 in Fig. 3B) when compared with the negative control (BF-MCESp). This indicates that TbISWI plays a role in maintaining a repressed chromatin structure around the ES promoter in T. brucei minichromosomes. The VSG Basic Copy arrays are another transcriptionally silent genomic location in T. brucei. Therefore, we also performed similar experiments using cell lines where eGFP reporter constructs (either with or without an ES promoter) were integrated in the VSG118 Basic Copy array (Fig. 2Bb, Fig 3C and Fig.

Fig. 4 ) 11 Page 14 of 40 results

 41140 . These data indicate that TbISWI plays a role in maintaining a repressed chromatin structure in both the T. brucei mini-chromosomes and VSG Basic Copy arrays in both bloodstream and procyclic form T. brucei. We can not exclude the possibility that the presence of an rDNA promoter directing transcription of the blasticidin resistance gene downstream of the reporter cassette could affect the local chromatin state. However, as the on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-are plotted as fold derepression (ratio of the mean fluorescence of induced cells to uninduced cells), the observed derepression can be inferred to be a consequence of TbISWI depletion.

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 1140 and 4B), we investigated whether there is a correlation between histone occupancy and TbISWI on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-in silent versus active VSG ESs. We used the bloodstream form T. brucei 221GPI(VO2+) line shown in (Fig. 4C), which has a neomycin resistance gene inserted behind the promoter of the active VSGVO2 ES (49). The silent VSG221 ES contains a puromycin resistance gene downstream of the ES promoter as well as a VSG pseudogene (ȥ). These are all single copy genes with the exception of VSGVO2, which has an additional copy in a VSG Basic Copy array. To determine the distribution of TbISWI over these silent and active VSG ESs, ChIP and qPCR analysis was performed. Interestingly, significant amounts of TbISWI (P<0.05;

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 1140 new data enabled us to re-investigate whether downregulation of TbISWI results in derepression of telomeric VSGs located in ESs, in addition to upregulation of the ES promoter proximal regions. The T. brucei 121-SA1 TbISWI RNAi cell line has an active VSG121 ES and contains eGFP downstream of the promoter of the silent VSG221 ES (21). Induction of TbISWI RNAi led to a decrease in TbISWI transcript, and a concurrent 60-fold increase of eGFP transcript (Fig. on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

VSGT3 11 Page 17 of 40 keeping

 1140 ES is active (data not shown). Although we detected VSG transcripts derived from the silent VSG ESs we did not detect VSG protein (result not shown), presumably as Western blot analysis is much less sensitive than qPCR. These data indicate that TbISWI could play a role in on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-the entire ES silenced. As Pol I transcribes VSG ESs, we next asked whether TbISWI is involved in silencing other Pol I-transcribed loci.

Fig. 2 ) 11 Page 18 of 40

 21140 . Flow cytometry was on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

on August 22 , 11 Page 19 of 40 results

 221140 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-indicate that TbISWI has a role in silencing all Pol I transcription units analysed: the VSG ESs, procyclin, as well as the rDNA spacer region in both life cycle stages.

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 1140 led us to hypothesize that TbISWI may play a role in establishing the epigenetic marks found on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

on August 22 , 11 Page 21 of 40

 221140 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-

  regions, constructs containing a small and genome context-sensitive ES promoter were integrated into these loci. Depletion of TbISWI resulted in 4 to 6-fold derepression of the exogenous ES on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-11

11 Page 23 of 40 able

 1140 , T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-to accommodate a single ES, this restriction could limit the amount of ES derepression observed after TbISWI depletion. TbISWI is involved in down-regulating Pol I transcription.

11 Page 24 of 40 transcription

 1140 mechanism. These observations all suggest that TbISWI is involved in regulation of on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-elongation in Pol I transcription units. This function of TbISWI is different to that seen in the regions containing silent VSGs in either the VSG Basic Copy arrays or minichromosomes. Here TbISWI presumably modulates chromatin structure, possibly preventing fortuitous initiation of transcription from cryptic promoters.

Ioc3p) downregulates transcription 11 Page 25 of 40

 1140 initiation[START_REF] Moreau | Regulated displacement of TBP from the PHO8 promoter in vivo requires Cbf1 and the Isw1 chromatin remodeling complex[END_REF], while the Isw1b complex (consisting of Isw1p in association with the Ioc4p and Ioc2p proteins) can facilitate both transcription elongation or termination[START_REF] Morillon | Isw1 chromatin remodeling ATPase coordinates transcription elongation and termination by RNA polymerase II[END_REF]. Additionally, in other organisms, different ISWI complexes have been shown to perform distinct functions in Pol I transcriptional control. The ISWI containing B-WICH complex is involved in activating Pol I transcription by promoting elongation[START_REF] Percipalle | Chromatin remodelling and transcription: be-WICHed by nuclear myosin 1[END_REF][START_REF] Percipalle | The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription[END_REF], whereas the ISWI containing NoRC complex is involved in repression of Pol I transcription and keeping the silent rRNA genes inactive[START_REF] Strohner | NoRC--a novel member of mammalian ISWI-containing chromatin remodeling machines[END_REF]. It is therefore possible that one TbISWI complex downregulates transcription elongation from Pol I transcription units, while another maintains a chromatin structure at the active loci which is facilitative to transcription.TbISWI is implicated in chromatin remodeling aroundPol II transcription units. on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-Recently in T. brucei and the related T. cruzi, acetylated and methylated histones were shown to be enriched in regions where polycistronic transcription units diverge, and which are proposed to be the sites of transcription initiation (40, 50, 63). The histone variants H2AZ and H2BV are also enriched in these regions. Convergent Pol II SSRs in T. brucei containing putative transcription termination sites are marked with histone variants H3v and H4v (50).

( 15 ) 11 Page 26 of 40 regionsonFig. 5 ononFig. 7

 15114057 Fig. 4

  Page 22 of 40 promoter in both BF and PF T. brucei. While it can be argued that this effect of TbISWI depletion is related to the ES promoter itself, the observation that levels of derepression from an ES promoter vary from about 6-fold in BF-MCESp1 or BF-VBESp1 to about 60-fold in a silent ES[START_REF] Hughes | A novel ISWI is involved in VSG expression site downregulation in African trypanosomes[END_REF] suggests that TbISWI-mediated repression of the ES promoter is dependent on genomic context. ES promoter derepression is not simply a consequence of a lethal phenotype, as RNAi mediated knock-down of another unrelated SNF2 domain containing protein, also led to a significant growth arrest but no observable ES derepression (Sup. Fig.10). It is likely that TbISWI is involved in preventing inappropriate transcription of the various pools of silent VSG genes, presumably by maintaining a repressive chromatin structure which plays a role in inhibiting fortuitous initiation of transcription. Our results are in agreement with studies from other eukaryotes, where ISWI chromatin remodelling complexes have been shown to be involved in transcription repression[START_REF] Corona | Multiple roles for ISWI in transcription, chromosome organization and DNA replication[END_REF][START_REF] Goldmark | The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p[END_REF][START_REF] Mellor | ISWI complexes in Saccharomyces cerevisiae[END_REF][START_REF] Ruiz | Cell-typedependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p-Itc1p chromatin remodelling complex[END_REF].

		Downloaded from
	Previously, we reported that TbISWI is important for silencing the promoters of the silent	http://ec.asm.org/
	VSG ESs, although it was unclear whether TbISWI depletion leads to processive transcription down to the telomeric VSG (21). Using new sequence data for T. brucei 427 VSG ESs (19), we re-investigated whether TbISWI plays a role in silencing telomeric ES located VSGs. After blocking TbISWI synthesis, we observed significant and reproducible increases in telomeric VSG transcripts. This implies that, contrary to the previous model, TbISWI has a role in keeping the entire silent ES inactive. The level of derepressed transcription from the silent ESs observed after TbISWI knock-down, was much lower than levels of transcription from an active ES	on August 22, 2018 by guest
	(discussed in (21)). The active ES is located in an Expression Site Body (ESB) (34), which is a
	subnuclear structure thought to contain a high concentration of Pol I as well as transcription and
	RNA processing factors essential for high levels of expression. As the ESB is thought to be only

Comment citer ce document : Stanne

on August 22, 2018 by guest http://ec.asm.org/ Downloaded from

Acknowledgements

We would like to thank Prof. Jane Mellor and David Clynes (Dept. of Biochemistry, University of Oxford) for advice about chromatin immunoprecipitation and ISWIs and allowing us to use the BioRuptor sonicator. We are grateful to Prof. Thomas Seebeck (University of Bern) for providing epitope tagging constructs, and Marcus Gould for some constructs. We thank Prof. Jay Bangs (University of Wisconsin) for providing anti-BiP antibody and Dr. Klaus Ersfeld for providing anti-SMC3 antibody. We thank Mani Narayanan, Megan Lindsay Povelones, Simone Wiesler, Marcin Dembek and other members of the Rudenko lab for discussions and comments on the manuscript. This work was supported by the Wellcome Trust. G.R. is a Wellcome Senior Fellow in the Basic Biomedical Sciences. M.K. is funded by the Inlaks Foundation and the Clarendon fund. on August 22, 2018 by guest http://ec.asm.org/ Downloaded from Version postprint Comment citer ce document : Stanne, T. M. (Co-premier auteur), Kushwaha, M. (Co-premier auteur), Wand, M., Taylor, J. E., Rudenko, G. (Auteur de correspondance) (2011). TbISWI regulates multiple polymerase I (Pol I)-transcribed loci and is present at Pol II transcription boundaries in Trypanosoma brucei. Eukaryotic Cell, 10 (7), 964-976. , DOI : 10.1128/EC.05048-11

Page 32 of 40 A representative slot-blot is shown for each probe.

While 100% of the DNA immunoprecipitated using an anti-myc antibody or no antibody was analysed on the slot-blots, only 0.1% of the input DNA and 1% of the anti-H3 immunoprecipitated DNA was analysed to avoid saturation and enable accurate quantitation. 6).

Fig. 2.

Schematic of the T. brucei reporter cell lines constructed for this study. (D) Distribution of TbISWI over silent and active VSG ESs. ChIP analysis was carried out on BF T. brucei cell lines expressing TbISWI (wt) and TbISWI-myc using Į-myc, and chromatin was immunoprecipitated as in (B). qPCR was used to quantitate amounts of genes shown in (C) (n=3 to 5 +/-SD). These genes are all single copy with the exception of VSGVO2, which has an additional copy present in a VSG Basic Copy array (BCA). Significant amounts of TbISWI were found binding both active and silent ESs (P<0.05; Sup. Table 8). 12). 13).


Clément Romano
email: clement.romano@cybel-llc.com@www.cybel-llc.com

Robert E Tench

Jean-Marc Delavaux

5W 1952nm Brillouin-Free Efficient Single Clad TDFA

Keywords: Single-frequency, fiber amplifier, Thulium, simulation, single clad, multistage, Brillouin

published or not. The documents may come

INTRODUCTION

Accurate simulations of TDFA is possible through fundamental characterization of the studied fibers used in a simple three level model of the rare-earth ion [START_REF] Romano | Characterization of the 3F4 -3H6 Transition in Thulium-doped Silica Fibres and Simulation of a 2μm Single Clad Amplifier[END_REF] . Such a modelization tool allows fast and efficient design of high performance amplifiers.

In previous publications we have demonstrated up to 2.6 W of signal output power out of a two stage single clad TDFA [START_REF] Tench | Broadband 2 W Output Power Tandem Thulium-doped Single Clad Fiber Amplifier at 2 μm[END_REF] . In this paper, we investigate a two stage SC TDFA that delivers a record 5 W of output power at 1952 nm without the presence of stimulated Brillouin scattering.

We have carried out our amplifier design by first optimizing the active fiber length of a single stage then followed by a SC booster stage. In that respect, we study the dependence of amplifier performance on slope efficiency, gain, and noise figure for both saturated and unsaturated conditions. Both co-and counter-pumped single stage amplifiers were experimentally and theoretically studied using two different commercial single clad Thulium-doped fibers (TDFs), which were from OFS and iXBlue. Using this study, we then have designed a two stage single clad TDFA capable of providing more than 5 W of saturated output power at 1952 nm. Comparisons of simulation and experimental data for the two stage SC TDFA are in good agreement.

ACTIVE FIBER LENGTH DEPENDENCY

In a recent publication [START_REF] Tench | Optimized design and performance of a shared pump single clad 2 µm TDFA[END_REF] , we showed with our in-house simulation software that varying the active fiber length of our TDFA allowed us to access different operating bands of the Thulium emission spectrum. In this section we confirm our modelization software by experimentally evaluating the dependence of the amplifier's performance on the active fiber length. We evaluated several amplifiers with different lengths of SC TDF in co-or counter-pumped configurations using either OFS TmDF200 or iXBlue IXF-TDF-4-125-v1. The pumping wavelength is ~1560 nm and the operating signal wavelength is either 1952 or 2004 nm. Signal and pump powers are measured at the input and output of the active fiber.

Performance of single stage using OFS fiber

We first investigated the dependence of the slope efficiency on fiber length for a single stage in a co-pumped configuration using the OFS fiber. The inset in Figure 1 shows the setup. Figure 1 shows the evolution of the slope efficiency (η) for an input signal power (Pin) of 2.1 dBm at 1952 nm as a function of the active fiber length (L). The slope efficiency is defined as the ratio of change in output signal power over change in pump power (η=ΔPout/ΔPpump). The experimental data (blue dots) compare very well with the simulation (blue line) that predicts a slope efficiency of 70 % for length above 2 m of active fiber. We note that the efficiency drops slightly with increasing fiber length from 70 % to 60 %. Our measured data indicate a slope efficiency of about 60 % for fiber length between 2 and 8 m. As we have shown in our evaluation of a tandem amplifier 3 the choice of fiber length determines the amplifier bandwidth of the TDFA. Figure 2 plots the normalized output spectra for a TDFA with 4 different active fiber lengths at the same input signal and pump power level. We define the bandwidth of the amplifier using a 10 dB criterion below the ASE peak for the amplifier in a saturated condition. As the fiber is shortened we observe that there is not much change in the gain at wavelength about 1952 nm. However the bandwidth shifts toward shorter wavelength and increases from 119 nm for 7 m to 134 nm for 1.5 m, and the peak gain wavelength shifts from 1930 nm for 7 m to 1845 nm at 1.5 m. The experimental and simulated spectral characteristics (ASE peak wavelength λpeak and the 10 dB bandwidth BW) are summarized in Table 1. All simulated spectra agree well with the measured data except for the fiber length of 1.5 m. For an active fiber length of 1.5 m the spectrum is cut off at short wavelength by the transmission bandwidth of the output isolator. The same co-pumped amplifier was then evaluated with an unsaturated input signal power (-20 dBm). The signal gain (G) and noise figure (NF) versus active fiber length (L) are shown in Figure 3. We measured a gain greater than 40 dB for length of active fiber greater than 3 m with a noise figure lower than 4 dB. The gain is found to be a slowly increasing function of the TDF length for lengths greater than 3 m, while the noise figure remains constant.

WDM

Performance of a single stage using iXBlue fiber

Next we evaluated the performance of a TDFA for both co-or counter-pumping configurations using the iXBlue single clad TDF. Figure 4 shows the slope efficiency versus active fiber length in a co-and counter-pumped configuration versus active fiber length for an input signal power of 2.7 dBm at 2004 nm. For both co and counter-pump configurations we measured a slope efficiency of 40 % for fiber length of about 4 m slightly lower than the simulated η results that indicates in co-pumping configuration 25 % and in counter-pumping configuration 50 %. The simulated η values is overestimated for the co-pumped configuration and underestimated for the counter-pumped configuration, the origin of this behavior is under study. Figure 6 plots the output spectra of the co-pumped iXBlue amplifier for different lengths of active fiber. As the fiber length increases the ASE is reabsorbed and its amplitude decreases, shifting the peak and the bandwidth to higher wavelengths.

Comparison of the experimental data (solid lines) and the simulation (dashed lines) shows a difference in the 1850-1950 nm wavelength range. This difference is under study. The OSNR does not change with the fiber length and is 59.8 dB/0.1 nm. The spectral characteristics of the iXBlue co-pumped amplifier are summarized in Table 2. We then study the gain and noise figure of the iXBlue single-clad amplifier in a counter-pumped configuration. Figure 7 shows the gain and noise figure of the amplifier for different input signal power versus the active fiber length. The amplifier has a gain higher than 25 dB and noise figure lower than 6 dB for an unsaturated input signal power. Good agreement is observed between the experimental data and the simulation. In comparison to the co-pumped configuration, the counterpumped amplifier delivers higher gain because of the higher pump power. In contrast the co-pumped amplifier pump power was limited by the onset of lasing. The output spectra of the amplifier in a counter-pumped configuration were measured and simulated for the different lengths studied and are displayed in Figure 8. Once again the OSNR does not change with the fiber length. The simulated and measured spectral characteristics are listed in Table 3. In comparison to the co-pumped configuration we observe a rather small change in the spectra as the fiber length increases. The ASE has a peak around 1860 nm for the experimental data and around 1850 nm for the simulation for all studied fiber length. As the λpeak, the 10 dB ASE bandwidth is not length dependent in a counter-pumping configuration. In a counter-pumping configuration most of the ASE is generated within the last few meters of the active fiber. Comparing the OFS and iXBlue single clad fibers we observed that for the tested configurations our simulation predicted the performance of the amplifier accurately. However, the simulated slope efficiency shows some difference with the measured values. In Table 4 have summarized the measured slope efficiency on single stage SC TDFA for both iXBlue and OFS fibers at two operating wavelengths. Both SC fibers exhibits a 60 % slope efficiency at 1952 nm whereas our simulation predicted about 75 %. At 2004 nm for the iXBlue fiber, the simulation underestimated slope efficiency in a copumping configuration and overestimated in a counter-pumping configuration. The difference of slope efficiency between the two wavelengths is attributed to the saturation power which is higher at 2004 nm than at 1952 nm. From our experimental evaluation of co-pumped amplifier, we noted that the 10 dB ASE bandwidth is large: 120 nm for the iXBlue fiber versus 130 nm for the OFS fiber. We remarked that the bandwidth increases with shorter fiber length, for both SC fibers. The bandwidth with the iXBlue fiber shows a stronger dependency on fiber length than with the OFS fiber. In contrast, the bandwidth of the iXBlue fiber in a counter-pumped configuration didn't change with respect to the active fiber length.

1800

λs (nm)

EXPERIMENTAL TWO STAGE AMPLIFIER CONFIGURATION

In our previous publications we have reported output power as high as 2.6 W for a multi-stage topology. In the next section we demonstrate a two stage amplifier where we increase the output power to more than 5 W.

The two stage amplifier topology is shown in Figure 9. Signal light from a single-frequency narrow linewidth discrete mode laser (DML) is coupled through an optical isolator into the first stage. This high gain stage is counter-pumped with a multi-watt ~1560 nm in-band fiber laser (P1). The fiber in the first stage (F1) is a 4.3 m long iXBlue fiber. Two configurations are considered depending on the inter-stage element: either the signal output of the first stage is coupled through an isolator (referred as TDFA-A), or through an ASE filter (referred as TDFA-B). The ASE filter is made of a circulator and a high reflection FBG centered at the seed wavelength. The second stage uses 2 m of iXBlue SC TDF as F2 which is co-pumped with a multi-watt pump (P2) at ~1560 nm. Input and output signal powers are measured internally at the input and output of the active fibers. Pump powers stated are the values coupled into the Thulium-doped fibers. An important consideration in high output power amplifier is the onset of Stimulated Brillouin Scattering, which limits the maximum output power. The linewidth of our single frequency input signal at 1952 nm was measured to be 7 MHz which is smaller than the fiber Brillouin gain bandwidth of 20 MHz 4 .

We consider the Brillouin to be generated and amplified into the active fiber F2. The output pigtail is too short to be considered (L<0.5 m). The Brillouin threshold ( SBS th P ) of the booster stage was estimated following [START_REF] Sincore | SBS Threshold Dependence on Pulse Duration in a 2053 nm Single-mode Fiber Amplifier[END_REF] Eq. 1 where  is the threshold parameter (21 u.a.), eff A is the optical mode area (12.6 µm²), eff L is the effective fiber length, and peak B g is the peak Brillouin gain coefficient (12.2 µm/W). eff A was calculated using the core diameter 4 µm and the NA 0.27 of the fiber given by the manufacturer. eff L was calculated using [START_REF] Lucas | 1 kW peak power, 110 ns single-frequency thulium doped fiber amplifier at 2050 nm[END_REF] Eq. 2 where out P is the output power of the active fiber,   Pz is the power profile along the fiber, and L the fiber length. The evolution of the signal power through the active fiber was given by our simulation tool. For an output power of 5 W the effective length of fiber was calculated to be 1.73 m. The SBS threshold was calculated to be 12.5 W, far above our output power goal.

EXPERIMENTAL AND SIMULATED RESULTS

Pre-amplifier stage performance

In out two stage TDFA, the first stage was designed to be a pre-amplifier focused on high signal gain at 1952 nm. Figure 10 plots the output signal power versus pump power for a saturating input signal. We demonstrate a slope efficiency of 62 % for the pre-amplifier in a saturated mode, delivering 1.4 W of output signal power for a pump power of 2.6 W. In order to prevent the onset of lasing and have a high input signal dynamic range, the pump power (Pp) was set to 1 W.

The measured and simulated gain (G) and the noise figure (NF) versus Pin are plotted in Figure 11. The pre-amplifier demonstrates a small signal gain greater than 35 dB with a noise figure lower than 7 dB. The simulation shows a difference with the measured data less than 1.5 dB. This pre-amplifier delivers gains higher than 35 dB for an unsaturated input signal combined with a noise figure lower than 7 dB and a high input dynamic range of 33 dB. These characteristics make this pre-amplifier highly suitable as an input for our booster stage.

Two stage amplifier performance

Following the evaluation of the pre-amplifier performance, its pump power was set to Pp=1 W in the two stage configuration in order to have an input signal dynamic range greater than 30 dB. Figure 12 plots the output power versus pump power for a saturated input signal for both TDFA-A & TDFA-B. We measured an output power greater than 5 W for both topologies with a slope efficiency η of 58 %, while our simulated slope efficiency is 78 %. The inset in Figure 12 shows the output spectra of both TDFA-A (in blue) and TDFA-B (in orange) for an output power of 5W at 1952 nm. The TDFA-A output spectra exhibits an OSNR of 53 dB with a 10 dB ASE bandwidth of 128 nm. In order to illustrate the power stability of TDFA-B the output power was set at 5 W and recorded over a 6 hour period.

The power stability was measured to be better than 2 % p-p showing no thermal effects in the amplifier. No SBS was observed for a single-frequency input signal at 1952 nm. Figure 14 plots the measured and simulated slope efficiency as a function of the input signal power for TDFA-A and TDFA-B. We measured a slope efficiency of 58 % for an input signal power greater than -10 dBm with TDFA-A. Even though TDFA-B introduces a higher inter-stage loss we clearly observe an improvement of the slope efficiency for Pin<-10 dBm of the booster stage over TDFA-A due to the ASE power generated by the pre-amplifier. In order to have a good power extraction (η = 58 %) within the second stage the input signal power should be greater than -20 dBm. The simulation shows a maximum slope efficiency of 80 %. This difference between the experimental and simulated η is under investigation. It could be caused by loss mechanisms not included in the simulation. We then compare the gain and noise figure versus Pin for both TDFAs in Figure 15. The results confirm that the addition of the ASE filter improves the amplifier dynamic range. Indeed, the input signal dynamic range improves to > 30 dB compared to the 15 dB with an inter-stage isolator only.

CONCLUSION

We have demonstrated a simple and robust two stage single clad amplifier delivering more than 5 W of output power at the 1952 nm signal wavelength. The performance of the two stage amplifier with two topologies were investigated: the first with an inter-stage isolator and the second with an inter-stage ASE filter. The ASE filter allowed us to demonstrate an input signal dynamic range greater than 30 dB with a slope efficiency in the booster stage of about 60 %. In addition a gain greater than 60 dB and NF lower than 7 dB were demonstrated. The output power stability at Pout=5 W was investigated and found to be better than 2 % over 6 hours. No Brillouin or other non-linear effects were observed with a single-frequency input signal.

The design of this two stage amplifier was guided by an experimental and theoretical study of single stage amplifier performance dependence on the active fiber length. The performance of the single stage amplifiers based on OFS TmDF200 or iXBlue IXF-TDF-4-125-v1 either co or counter-pumped were investigated. The comparison between experimental and simulated gain, noise figure, output power and slope efficiency shows overall in good agreement.

Since the two stage amplifier output power was not limited by the available pump power, we should be able to scale the amplifier to higher output power. In the future, we intend to investigate other single clad fibers for the booster stage to improve the efficiency.

Figure 1 :

 1 Figure 1: Slope efficiency versus active fiber length for the OFS fiber in a co-pumped configuration.

Figure 3 :

 3 Figure 3: Gain and noise figure versus OFS fiber length for an unsaturated input.

Figure 4 :

 4 Figure 4: Slope efficiency versus iXBlue fiber length in a co-and counter-pumped configuration.

Figure 5

 5 Figure5shows the performance of an amplifier in a co-pump configuration for three fiber lengths: 3.3, 4.3, and 5.3 m. The experimental and simulated results agree well for Pin of -9, -19 and -29 dBm and yield a gain greater than 20 dB. The noise figure measurements are in agreement with the simulation results. We observe almost no dependency of gain and noise figure to the active fiber length between 3 m and 7 m.

Figure 5 :

 5 Figure 5: Gain and noise figure of the iXBlue co-pumped amplifier versus active fiber length.

Figure 7 :

 7 Figure 7: Gain and noise figure of the iXBlue counter-pumped amplifier versus active fiber length.

F2Figure 9 :

 9 Figure 9: Topology of our two stage TDFA operating at 1952 nm.

Figure 10 :

 10 Figure 10: First stage output power versus pump power at 1952 nm.

Figure 11 :

 11 Figure 11: First stage gain and noise figure versus input signal power at 1952 nm.

Figure 12 :

 12 Figure 12: Two stage amplifier output signal power versus pump power for both TDFAs.

Figure 13 :

 13 Figure 13: Output power stability of TDFA-B for a saturating input signal.

  Figure 14: Two stage slope efficiency versus input signal power for both TDFAs.

Figure 15 :

 15 Figure 15: Gain and noise figure versus input signal power for TDFA-A and TDFA-B.

L (m) Measured Simulated λpeak (nm) BW (nm) λpeak (nm) BW (nm)

		0	OFS Co-pumped
	Output relative spectrum (dB)	-60 -40 -20	P1 P in =2.1 dBm @  s =1952 nm =1 nm P p =2.6 W @  p =1567 nm		TDF, OSNR=48 dB/1 nm L L (m) Sim 7 5 3 1.5	Data
			P			P
			in			out
		-80
					1900	2000	2100
					Wavelength (nm)
	Figure 2: Measured and simulated spectra of a co-pumped amplifier for different OFS fiber lengths.
			1.5	1845	134	1849	143
			3	1895	134	1889	128
			5	1915	124	1913	117
			7	1930	119	1926	112

Table 1 :

 1 Experimental and simulated ASE characteristics of the OFS co-pumped amplifier.

Table 2 :

 2 Experimental and simulated ASE characteristics of the iXBlue co-pumped amplifier.

		0
			L (m) Data Sim
			3.3
			4.3
	Relative output power (dB)	-40 -20	5.3	iXBlue Co-pumped P p =2.5 W @  p =1560 nm	OSNR=59.8 dB/0.1 nm
		-60		P in =2.7 dBm @  s =2004 nm
				=1 nm
				1900		2000	2100
					Wavelength (nm)
				Measured	Simulated
			L (m)
				λpeak (nm)	BW (nm)	λpeak (nm)	BW (nm)
			5.3	1908	123	1931	134
			4.3	1897	117	1920	130
			3.3	1887	96	1902	133

Figure 6: Output spectrum of the iXBlue co-pumped amplifier for different active fiber length.

Table 3 :

 3 Figure 8: Output spectrum of the iXBlue counter-pumped amplifier for different active fiber length. Experimental and simulated ASE characteristics of the iXBlue counter-pumped amplifier.

		0
			L (m) Data Sim
			3.3
			4.3
				OSNR=53.8 dB/0.1 nm
	Output relative power (dB)	-40 -20	5.3	iXBlue Counter-pumped P p =2.5 W @  p =1560 nm
		-60		P in =2.7 dBm @ 	s =2004 nm
				=1 nm
		1700	1800	1900	2000	2100
				Wavelength (nm)

Table 4 :

 4 Measured slope efficiency of both SC TDF at two operating wavelengths for Pin≈2 dBm

		2004
	TDF	η (%)
	OFS	63	/
	iXBlue	62	44

ACKNOWLEDGEMENTS

We thank iXBlue for the single clad TDF, and the FBGs, OFS for the single clad TDF, and Eblana Photonics for the singlefrequency DML sources around 2 µm.