Drosophila Kismet Regulates Histone H3 Lysine 27 Methylation and Early Elongation by RNA Polymerase II

Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation—a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin

Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Funder: Studienstiftung des Deutschen VolkesGermline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions

Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs

The <em>Drosophila</em> Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics <em>In Vivo</em>

<div><p>dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in <em>Drosophila</em>. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.</p> </div

The over-expression of dMi-2 does not decrease cohesin levels.

<p>(A) Protein blot of salivary gland chromatin extracted from <i>UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts</i> individuals raised at 18°C until the late third-instar stage and then shifted to 29°C for 24 hours to induce <i>UAS-Mi-2⁺</i> expression. The blot was probed with antibodies against Smc1 and histone H3 as a control. (B, C and D) RT-PCR analysis of Smc1, SA, and Rad21 RNA levels in the salivary glands of <i>UAS-LacZ/+</i>; <i>da-GAL4/+</i> (<i>UAS-LacZ</i>) control larvae and <i>UAS-dMi-2⁺ 3-3/+</i>; <i>UAS-dMi-2⁺ 15-1/da-GAL4</i> (<i>UAS-Mi2⁺</i>) larvae raised at 29°C. Histone H1 RNA levels are shown as a control.</p

dMi-2 colocalizes with cohesin.

<p>(A–C) Magnified images of salivary gland polytene chromosomes stained with antibodies against dMi-2 (red) and Pol II Ser2 (A, green), stromalin (B, green) and Nipped B (C, green). Note the extensive overlap between the chromosomal distributions of the four proteins. (D) Reducing <i>dMi-2</i> gene dosage suppresses the small wing blade phenotype of individuals heterozygous for the <i>Nipped-B⁴⁰⁷</i> null allele. A minimum of twenty adult male wing blade areas was measured for each of the indicated genotypes, and the distributions of blade areas are presented as box-plots. For each genotype, the chromosome to the left of the separator (/) came from the male parent, and the chromosome to the right came from the female parent. The P57B chromosome is the wild-type chromosome in which the <i>Nipped-B⁴⁰⁷</i> mutation was induced by γ rays <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002878#pgen.1002878-Rollins1" target="_blank">[39]</a>. For the +/<i>Nipped-B⁴⁰⁷</i>, +/<i>dMi-2⁴</i> and +/P57B genotypes, the wild-type chromosomes came from an Oregon R male parent.</p

Analysis of GAL4-regulated transgenes.

<p>Data are shown for progeny of the following crosses:</p>(a)<p><i>dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(b)<p><i>w; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; dMi-2⁴ da-GAL4/TM6B</i>, <i>Tb</i> females;</p>(c)<p><i>w; P[w⁺, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5 males X <i>w; da-GAL4</i> females;</p>(d)<p><i>w; P[w⁺, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5; dMi-2^f08103/TM6B, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(e)<p><i>w; P[w+, UAS-dMi-2</i>^{Δ<i>932-1158</i>}]6-5; dMi-2⁴/TM6B, <i>Tb</i> males X <i>w; da-GAL4</i> females;</p>(f)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; dMi-2⁴ da-GAL4/TM6B</i>, <i>Tb</i> females;</p>(g)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3</i> males X <i>w; da-GAL4</i> females;</p>(h)<p><i>w; P[w⁺, UAS-dMi-2⁺]3-3; dMi-2⁴/TM6B</i>, <i>Tb</i> males X <i>w; da-GAL4</i> females.</p

dMi-2 is required for full decondensation of heat-shock loci.

<p><i>w¹¹¹⁸</i> control and transgenic larvae expressing dominant-negative dMi-2^K761R were subjected to a 20 min heat-shock at 37°C. Polytene chromosomes were visualized by staining with DAPI (right panels) or indirect immunofluorescence using an antibody against Pol II Ser2 (left panels). Arrows indicate the <i>hsp70</i> gene containing loci 87A and 87C.</p

dMi-2 does not promote chromatin decondensation by antagonizing histone H1 assembly.

<p>(A–B) Live analysis of salivary gland nuclei of late third-instar <i>UAS-LacZ/+; ey-GAL4/+</i> (UAS-LacZ) control larvae (A) and <i>UAS-dMi-2⁺ 3-3/+</i>; <i>UAS-dMi-2⁺ 15-1/ey-GAL4</i> (<i>UAS-dMi-2⁺</i>) larvae (B) expressing H1-GFP. Scale bars are 10 µm. (C) Quantification of H1-GFP fluorescence in larvae shown in A and B. The exposure times used to capture the images are identical; the number of glands analyzed is noted. (D) Protein blot showing the relative levels of histones H1 and H3 in chromatin extracted from late third-instar <i>UAS-dMi-2⁺ 3-3/+</i>; <i>da-GAL4/+</i> (<i>UAS-dMi-2⁺</i>) and <i>UAS-LacZ/+</i>; <i>da-GAL4/+</i> (<i>UAS-LacZ</i>) larvae raised at 29°C.</p