AHMAD LAB

WELCOME TO THE AHMAD LAB

Dissecting chromatin function

We focus on how chromatin structure and dynamics affect gene regulation and transcription. Eukaryotic genomes are packaged into nucleosomes, where DNA is tightly wrapped around octamers of core histones. Histones and nucleosome structure are extremely conserved throughout eukaryotes, and these nucleosomes are formidable barriers to DNA-binding factors and the progression of RNA polymerases. Cells devote considerable effort to manipulating nucleosomes to control transcription. We explore this regulation using model developmental systems in Drosophila.

 

Nucleosome dynamics in active chromatin

file

Although the organization of chromatin appears static in genomes, the processes of gene activation, transcription, and chromatin remodeling drive continual changes in structure. These changes served to fine-tune the exposure of transcription factor binding sites, and clear the path for elongating RNA polymerases. Structural changes to nucleosomes include pushing nucleosomes around, partially unwrapping nucleosomes, and at its most extreme stripping histones off of DNA. These processes require histone chaperones and chromatin remodelers to both dis-assemble old nucleosomes and then re-assemble new ones.

Our studies of nucleosome dynamics were initially stimulated by studying histone variants encoded in the Drosophila genome. Alternative histone variants are conserved throughout eukaryotic evolution, and we found that one of these – the H3 histone variant H3.3 – is the exclusive substrate for a distinct nucleosome assembly system. This Replication-Independent (RI) system is dedicated to rebuilding nucleosomes in dynamic chromatin regions, particularly at transcribed genes. The RI assembly system and its’ use of H3.3 is now known to be conserved in all eukaryotes. We currently study the factors that mediate RI assembly, and the importance of RI assembly and H3.3 for chromatin function. Recent discoveries with human patients highlight the importance of H3.3: some ‘driver’ mutations in aggressive cancers map to both the H3.3 histone and in H3.3-specific assembly factors. We use Drosophila to define how these mutations affect chromatin function.

For more:

JI Schneiderman, GA Orsi, KT Hughes, B Loppin, & K Ahmad (2012). Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant. PNAS USA 109:19721-19726.

A Sakai, BE Schwartz, S Goldstein, & K Ahmad (2009). Transcriptional and developmental functions of the H3.3 histone variant in Drosophila. Current Biology 19:1816-1820.

K Ahmad & S Henikoff (2002). Epigenetic consequences of nucleosome dynamics. Cell 111:281-284.

K Ahmad & S Henikoff (2002). The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Molecular Cell 9:1191-200.
 

Polycomb-mediated silencing during development

Eukaryotes also devote substantial effort to gene silencing. Polycomb silencing is a classic system of chromatin-based silencing; it was first identified in Drosophila, but is a conserved aspect of establishing and maintaining developmental programs in multicellular eukaryotes. Polycomb-regulated chromatin domains have characteristic histone modifications, and many of the enzymes, chromatin proteins, and RNA components in this system are now defined. However, how Polycomb interacts with developmental programs remains poorly understood. Our research focuses on the following questions: How do developmentally-regulated enhancers integrate with Polycomb silencing? What is the mechanism of silencing? How do cells switch between silenced and active chromatin states? How is the Polycomb-silenced state epigenetically inherited?

file

We use new high-resolution chromatin mapping methods to characterize developmental changes in the epigenome. The MNase-seq method comprehensively maps both nucleosomes and bound transcription factors in native chromatin, with base-pair resolution. A second method – native Chromatin Immunoprecipitaton (native-ChIP) – recovers and maps specific chromatin-bound factor complexes. MNase-seq and native-ChIP empirically define the sequences that are protected by transcription factors in a cell type. Our overall goal is to define the motif grammars and interactions that determine factor occupancy and control regulatory elements throughout the genome.

Classic work in Drosophila defined short regulatory elements called Polycomb Response Elements (PREs) within domains that are essential for Polycomb silencing. PREs are thought to nucleate the binding of PRC1 and PRC2 complexes to nearby nucleosomes. However, using MNase-seq and native-ChIP we discovered that Polycomb is anchored to PREs through specific transcription factors. We identified a set of conditional binding sites where transcription factors and Polycomb only bind in the repressed epigenetic state; in other cell types where the domain is active, occupancy and size of bound complexes at the PRE is reduced. At other sites Polycomb is constitutively bound; these sites are often the promoters of developmentally-regulated genes. Constitutive Polycomb binding may mark potential target promoters, and binding at PREs may switch domains between chromatin states. The ability of PREs to switch states between cell types seems to require exceptionally poor factor-binding motifs, which we suspect is a design feature of developmentally-responsive regulatory elements. We are testing these ideas using the vestigial gene as a model Polycomb-regulated domain in Drosophila, where the developmental enhancers of the vestigial gene are known. Thus, at the vestigial gene we can define how positively-acting elements interact with silencing elements in Polycomb-regulated domain.

In a genome-wide perspective, we are characterizing the changes in Polycomb-regulated domains during the specification and differentiation of the Drosophila mesoderm. Our goal is to define how Polycomb contributes to a complete developmental program.

For more:

GA Orsi, S Kasinathan, KT Hughes, S Saminadin-Peter, S Henikoff, & K Ahmad (2014). High-resolution mapping defines the cooperative architecture of Polycomb Response Elements. Genome Research 24:809-20. PMCID: PMC4009610.

S Kasinathan, GA Orsi, GE Zentner, K Ahmad, & S Henikoff (2014). High-resolution mapping of transcription factor binding sites on native chromatin. Nature Methods 11:203-209.

 

Chromatin epigenetics

Every once in awhile we stumble across surprising epigenetic phenomena. Some of these reveal the capability of the nucleus to control allelic expression, to clonally inherit gene expression patterns, and to copy nucleosome patterns. We use these to study how epigenetic information is stored in chromatin. We are particularly interested in testing mechanisms of chromatin inheritance.

file

For more:

F Greil & K Ahmad (2012). Nucleolar dominance of the Y chromosome in Drosophila melanogaster. Genetics 191:1119-28. PMCID: PMC3415996.

JI Schneiderman, S Goldstein, & K Ahmad (2010). Perturbation analysis of heterochromatin-mediated gene silencing and somatic inheritance. PLoS Genetics 6:e1001095. PMCID: PMC2936522.