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1.
Figure 3

Figure 3. UpSET interacts with an HDAC complex. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A) UpSET-bound regions are enriched for the Histone deacetylase Rpd3 and its associated cofactor Sin3. Box plot analysis of Rpd3 and Sin3 DamID signal overlapping with UpSET bound regions.
(B) Browser view of chromosome 2L highlighting the overlapping DamID signal for UpSET, Rpd3 and Sin3.
(C) Venn diagram showing the overlap among regions bound by UpSET, Rpd3, and Sin3. UpSET overlaps with only half of the loci co-bound by Rpd3 and Sin3.
(D–G) UpSET co-localizes with Sin3 and Rpd3 on chromosomes. Polytene chromosome sets stained with a mixture of antibodies to NH3- UpSET (D’-D”, E’-E”, F–G; green) and Rpd3 (D, D”, G; red) or Sin3 (E, E”, F; red). DNA was counterstained with DAPI (blue). (F–G) Higher magnification views showing co-localization of UpSET with Rpd3 (G) or Sin3 (F).
(H) UpSET interacts with Rpd3 and Sin3. Co-immunoprecipitation of UpSET with Rpd3 and Sin3 from Kc nuclear extracts. Arrows indicate the specified protein. 20% input is shown.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.
2.
Figure 2

Figure 2. UpSET is preferentially recruited to active genes. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A) Sites of UpSET recruitment based on DamID. The black vertical lines represent the relative position on the chromosomes of the ~14000 Drosophila genes. Blue dots represent single peaks (3.5-6Kb) and the red dots (>6 Kb) UpSET binding sites.
(B–C) UpSET chromatin profile over a 600Kb region of chromosome 3R and a 11Kb region of chromosome 2L. Single peaks and regions are presented in blue and red, respectively. The mean signal was smoothed + whiskers. Coding regions are shown in blue (B) and black (C).
(D) UpSET is mainly enriched in promoter (TSS) regions. Box plots of the UpSET DamID-based chromatin profile over the 9 different chromatin states reported by the modENCODE Consortium. TSS: transcriptional start sites; Exon: elongating regions; Enh: Enhancers; OC: open chromatin; MXC: male X genes; Pc: Polycomb; HC: constitutive heterochromatin; HLC: heterochromatin-like in euchromatin; IEC: basal, intergenic euchromatin. Random: set of 6000 random sequences. *: p< 2.2X10−16
(E) Preferential association of UpSET over transcribed genes. End analysis of the UpSET chromatin profile to the 5’ and 3’ end of genes ranked by expression quintiles from high (Q1) to low (Q5) gene expression.
(F) End analysis of UpSET DamID signal to RNA Pol II (black) and H3K4m3 (red) enriched regions. See also Figure S2.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.
3.
Figure 6

Figure 6. UpSET modulates developmentally regulated gene silencing. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A–F) upSET mutants show incomplete or fused egg chambers (arrows) in comparison to wildtype. Wildtype (A–C) and upSETe00365 mutant (D–F) ovarioles stained with DAPI (blue; to visualize DNA) and phalloidin (white). Confocal projections of 5 serial 1μm optical sections are shown.
(G–I) UpSET is required for the establishment of anterior-posterior axis asymmetry and proper egg chamber formation. Wildtype (G) or upSETe00365 mutant (H–I) ovarioles stained with the oocyte-specific marker Orb showing alterations on the oocyte position and improper pinching off of egg chambers (arrowhead) from the germarium. Confocal projections of 8 serial 1μm optical sections are shown.
(J–K’) UpSET protein levels are differentially regulated during oogenesis. Wildtype ovarioles were stained with a mix of antibodies against UpSET NH3- and COOH- termini and co-stained with DAPI (blue). Confocal projections of 5 serial 1μm optical sections are shown. Arrowhead indicates regions with differential UpSET levels.
(L–M) Wildtype (L) and upSETe00365 mutant (M) ovaries stained with antibodies against the Notch intracellular domain (green) and DAPI (blue).
(N–O) Co-staining of SOCS36 (green) and Eya (red) in wildtype (N) and upSETe00365 mutant (O) ovarioles. DAPI (blue) was used to visualize DNA. Overlapping expression is indicated with an arrowhead in upSET mutant ovary.
(P) UpSET restricts histone acetylation and chromatin accessibility around promoter/genic regions via interaction and stabilization of Rpd3/Sin3-containing HDAC machinery. Lack of UpSET increases chromatin accessibility that correlates with a higher histone acetylation level due to the loss of Rpd3 from transcriptionally active genes. Changes in the chromatin landscape increase the probability of activating transposon expression and off-target genes.
Posterior is oriented to the right in all images. Scale bars represent 50μm.
See also Figure S7.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.
4.
Figure 5

Figure 5. UpSET modulates off-target gene. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A–B) UpSET knock down up-regulates silent genes. Gene expression analysis of knock down Kc cells using primers (W–Z) targeting the genomic region from Figure 4F, as well as the coding sequence of the UpSET target gene CG4389. (*: p <0.05; **: p<0.01; Not significant p values are not marked).
(C) Non-expressed genes are more affected upon UpSET knock down. Genes differentially expressed in UpSET knock down cells were clustered by their association with RNA polymerase II.
(D–E) Transcriptome analysis of upSETe00365 mutant ovaries. Heat maps show the summary of three biological replicates of independent upSETe00365 mutant ovaries compared to wildtype (D). Up-regulated (red) and down-regulated (green) genes are shown. Gene ontology analysis of the differentially expressed genes (E). 22 categories were enriched in the dataset (p <0.001).
(F) RNA-seq from wildtype ovaries (modENCODE) was clustered in gene expression quintiles from high (Q1) to low (Q5) and upSETe00365-affected gene percentage for each quintile is shown.
(G) Low/silent genes are up-regulated in upSETe00365 mutants. RT-qPCR of low expressing genes (Q4-5) was compared to highly expressed genes (Q1). (*: p <0.05; **: p<0.01; Not significant p values are not marked).
(H–K) Loss of UpSET increases expression of transposons. RT-qPCR of transposons from 9 representative repetitive element families found near UpSET binding sites in upSETe00365 mutant ovaries (H) or UpSET knock down Kc cells (K). cDNA tiling analysis of transposon expression in upSET e00365 ovaries with DamID chromatin profile for a single peak (I) or region (J) of UpSET binding (see also Figure S2A). The middle track shows mRNA expression in ovaries. Repeat element locations are shown in lower panel. Bars represent mean +/− SEM. (*: p <0.05; **: p<0.01; Not significant p values are not marked)
See also Figure S6.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.
5.
Figure 1

Figure 1. UpSET is a SET-containing nuclear protein required for Drosophila oogenesis. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A) Schematic of the upSET locus on chromosome 3L. Location of the SET and PHD domains are highlighted. Arrowhead indicates P-element insertion in upSETe00365. Graph shows the locus conservation in the Drosophilidae family.
(B) UpSET shares only ~15% identity with mouse MLL5 in the SET domain. The mouse MLL5 SET domain shares 60% identity with that of mouse SETD5.
(C) Sequence alignment of the SET and PHD domains between Drosophila UpSET and mouse MLL5. % identity is indicated.
(D) UpSET is mainly a nuclear protein. Western blot analysis of nuclear (NE) and cytoplasmic (Cyt) extracts from Kc cells probed with anti-UpSET N-end monoclonal antibodies. Full-length UpSET protein is denoted by the arrow. *:Non-specific band.
(E–E’) UpSET forms foci in the nucleus. Confocal projection of 10 serial 0.2μm optical sections of a Kc cell stained with a mix anti-UpSET NH3-terminus monoclonal antibodies (green; F–F’) and DAPI to visualize DNA (F’).
(F–H) Third larval instar polytene chromosomes stained with a mix of antibodies to UpSET NH3- and COOH-terminus (green) and DNA counterstained with DAPI (blue). Higher magnification view of X chromosome (H). UpSET recruitment is enriched in euchromatic regions (I).
(I) UpSET does not exhibit histone methyltransferase activity (HMT). Radioactive HMT assays performed with bacterially purified GST or GST-UpSET SET domain and purified histones as substrate (J). Recombinant proteins were incubated with Super Sex Combs (SXC) or nuclear extracts (NE). SET7 was used as positive control. Loading controls for the histone methyltransferase assay are shown.
(K) Polytene chromosome from upSETe00365 mutant third instar larva stained with a mix of the 5 UpSET monoclonal antibodies (green) and DNA counterstained with DAPI (blue).
(J) Western blot analysis of larval nuclear extracts from wildtype and upSETe00365 mutants blotted with a mix of the 5 antibodies against UpSET NH3- and COOH-termini. Lamin Dm0 serves as loading control.
(L–M) Lack of UpSET disrupts oogenesis. Confocal projections of 5 serial 1μm optical sections of ovaries from wildtype (N) and upSETe00365 mutants (O) actin stained with phalloidin. Scale bars represent 100μm. See also Figure S1.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.
6.
Figure 4

Figure 4. UpSET modulates active chromatin modifications of transcriptionally active genes. From: UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions.

(A) RNAi-based knock down of UpSET levels. Western blot for UpSET from nuclear extracts of Mock and UpSET-specific RNAi expressing cells same antibodies as in Figure 1J. RNA Pol II was used as loading control.
(B) UpSET knock down increases histone acetylation over UpSET bound promoters (TSS). Native ChIP of H3Ac, H3K9Ac and H4K16Ac from Mock and UpSET knock down cells as indicated. Data are represented as mean +/− standard error of the mean (SEM). N: Notch; Rel; Relish; Neur: neuralized; Dl: Delta; Orb: oo18 RNA-binding protein; Su(H): Suppressor of Hairless, and the PcG-silenced C15 gene, as negative control. (*: p<0.05; **: p<0.01; ***: p<0.001, non-significant p values are not marked).
(C–D) Cross-linked ChIP for Rpd3 and Sin3 from Mock and UpSET knock down cells. Bars represent mean +/− SEM.
(E–F) Histone acetylation increases around UpSET-depleted genes. Native ChIP of H3Ac, H3K9Ac and H4K16Ac from Mock and UpSET knock down cells as indicated. UpSET chromatin profiles show the location of the used primers. Repressive chromatin reported from (Bartkuhn et al., 2009); blue line). Bars represent mean +/− SEM.
(G–H) Native ChIP of H3K4m1, H3K4m2, H3K4m3 and H3K27m3 from Mock and UpSET knock down cells. Primers are same as in Figure 4E–F. Bars represent mean +/− SEM. (*: p<0.05; **: p<0.01; ***; p<0.001).
(I) UpSET depletion increases the accessibility of UpSET bound regions. Chromatin accessibility ratio in KD:mock treated cells from a 150Kb region of chromosome 2L.
(J) UpSET associated regions show increased accessibility. Chromatin accessibility signal distributions over UpSET bound regions or random sites. p<2.2x10−16
(K) UpSET modulates chromatin accessibility of transcriptionally active genes. End analysis of the chromatin accessibility signal in UpSET knock down cells over the 5’ and 3’ ends of genes clustered by expression quintiles from high (Q1) to low (Q5) gene expression as indicated.
(L–P) Chromatin accessibility in situ. Schematic of M.SssI DNA methyltransferase accessibility assay in ovaries. Low CpG methylation as detected by 5mC immunofluorescence in untreated Drosophila germarium from wildtype (M) and upSETe00365 mutants (N). Higher chromatin accessibility in M.SssI-treated upSETe00365 mutant ovaries (P) compared to wildtype (O). See also Figures S3-S5.

Hector Rincon-Arano, et al. Cell. ;151(6):1214-1228.

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