Features of Cryptic Promoters and Their Varied Reliance on Bromodomain-Containing Factors

The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters

H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function

The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs

A Whole-Chromosome Analysis of Meiotic Recombination in Drosophila melanogaster

Although traditional genetic assays have characterized the pattern of crossing over across the genome in Drosophila melanogaster, these assays could not precisely define the location of crossovers. Even less is known about the frequency and distribution of noncrossover gene conversion events. To assess the specific number and positions of both meiotic gene conversion and crossover events, we sequenced the genomes of male progeny from females heterozygous for 93,538 X chromosomal single-nucleotide and InDel polymorphisms. From the analysis of the 30 F1 hemizygous X chromosomes, we detected 15 crossover and 5 noncrossover gene conversion events. Taking into account the nonuniform distribution of polymorphism along the chromosome arm, we estimate that most oocytes experience 1 crossover event and 1.6 gene conversion events per X chromosome pair per meiosis. An extrapolation to the entire genome would predict approximately 5 crossover events and 8.6 conversion events per meiosis. Mean gene conversion tract lengths were estimated to be 476 base pairs, yielding a per nucleotide conversion rate of 0.86 × 10−5 per meiosis. Both of these values are consistent with estimates of conversion frequency and tract length obtained from studies of rosy, the only gene for which gene conversion has been studied extensively in Drosophila. Motif-enrichment analysis revealed a GTGGAAA motif that was enriched near crossovers but not near gene conversions. The low-complexity and frequent occurrence of this motif may in part explain why, in contrast to mammalian systems, no meiotic crossover hotspots have been found in Drosophila

Composition and Function of Mutant Swi/Snf Complexes

Summary: The 12-subunit Swi/Snf chromatin remodeling complex is conserved from yeast to humans. It functions to alter nucleosome positions by either sliding nucleosomes on DNA or evicting histones. Interestingly, 20% of all human cancers carry mutations in subunits of the Swi/Snf complex. Many of these mutations cause protein instability and loss, resulting in partial Swi/Snf complexes. Although several studies have shown that histone acetylation and activator-dependent recruitment of Swi/Snf regulate its function, it is less well understood how subunits regulate stability and function of the complex. Using functional proteomic and genomic approaches, we have assembled the network architecture of yeast Swi/Snf. In addition, we find that subunits of the Swi/Snf complex regulate occupancy of the catalytic subunit Snf2, thereby modulating gene transcription. Our findings have direct bearing on how cancer-causing mutations in orthologous subunits of human Swi/Snf may lead to aberrant regulation of gene expression by this complex. : Subunits of the Swi/Snf chromatin-remodeling complex are mutated in 20% of cancers. Dutta et al. report modularity within yeast Swi/Snf that regulates both architecture and genomic functions of the complex. These findings will help predict complex composition in diseased states where subunits of the complex are mutated. Keywords: modularity, architecture, Swi/Snf complex, patterns, gene expression, targeting, Snf2 occupancy, Swi/Snf subunits, residual Swi/Snf complexes, cance

Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression

Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription

Global Analysis of H3K4 Methylation Defines MLL Family Member Targets and Points to a Role for MLL1-Mediated H3K4 Methylation in the Regulation of Transcriptional Initiation by RNA Polymerase II ▿ †

A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. Set1/COMPASS was the founding member and is the only H3K4 methylase in Saccharomyces cerevisiae; however, in mammals, at least six H3K4 methylases, Set1A and Set1B and MLL1 to MLL4, are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation patterns in wild-type Mll1+/+ and Mll1−/− mouse embryonic fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes, show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/MLL4 and/or the Set1 complexes has little to no effect on the H3K4 methylation of Hox loci or their expression levels in these MEFs. Together these data provide insight into the redundancy and specialization of COMPASS-like complexes in mammals and provide evidence for a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation