Welcome to the Bartholomew Lab!
The Bartholomew laboratory has developed techniques for probing protein-protein and protein-DNA interactions to understand the architecture of chromatin remodeling complexes, how they interact with nucleosomes, and the structural changes associated with chromatin remodeling. The lab has exploited this information to dissect the functional roles of different domains and subunits in nucleosome remodeling using targeted mutagenesis combined with both state-of-the-art next-generation sequencing approaches and detailed biochemical assays. The lab's primary focus is on how these remodelers shape the chromatin structure at promoter and enhancer regions and regulate formation of preinitiation transcription complexes, release of paused RNA polymerase, and transcription directionality. The future objectives are to understand how these remodelers control the synthesis of messenger RNA, non-coding RNA, and anti-sense RNA arising from divergent transcription at promoter regions. In addition, the lab seeks to understand the roles of ATP-dependent chromatin remodelers in nuclear organization and promoting long-range chromatin interactions using chromatin capture-based approaches coupled with other techniques to physically map these interactions.
The yeast SWI/SNF chromatin remodeling complex regulates the synthesis of non-coding RNA
SWI/SNF is one of the most frequently mutated epigenetic factors in
cancer, with mutations occurring in ~20-25% of all cancers. Mutations
in SWI/SNF are also the molecular drivers of several neurological
disorders. The Bartholomew laboratory is now defining the functional
roles of specific highly conserved SWI/SNF protein domains in the
catalytic subunit and other accessory subunits, using yeast as a
model. One such domain is the AT hook domain, which directly contacts
both the SWI/SNF ATPase and SnAC regulatory domains. The lab has now
shown that the AT hook is important for positively regulating SWI/SNF
chromatin remodeling activity. Although the AT hook has a high
affinity for AT-rich DNA sequences, unexpectedly, the Bartholomew lab
found that this domain is required neither for SWI/SNF to efficiently
bind nucleosomes nor to be recruited by transcriptional activators.
However, by using MNase-seq, a next-generation sequencing-based
genomic approach, the laboratory showed that loss of the AT hook
reduced nucleosome positioning at the -1 and +1 sites, which flank
transcriptional start sites, and RNA-seq experiments indicated that
the AT hook contributes to the ability of SWI/SNF to suppress
anti-sense transcription upstream of coding regions. Together, this
shows SWI/SNF has roles in both promoting nucleosome positioning at
promoters and repressing anti-sense transcription.
The mammalian SWI/SNF complex in pluripotency and development
Guided by their findings in yeast, The Bartholomew lab is now
examining the function of the SWI/SNF complex (i.e. esBAF) in mouse
embryonic stem cells (mESCs). SMARCA4/BRG1 is the catalytic subunit of
the esBAF complex and is required for both maintenance and
proliferation of stem cells as well as for stem cell exit from
pluripotency and subsequent differentiation. By deleting the AT hook
region from both copies of Smarca4 using CRISPR-Cas9, the lab
has found that the AT hook is required for exiting, but not for
maintaining pluripotency. By mutationally uncoupling these activities
and then performing PRO-seq, a method to map transcribing RNA
polymerase II (RNAPII) at base pair-resolution, the Bartholomew lab
found that the esBAF complex released RNAPII pausing at developmental
genes as the cells transitioned from their ground/naïve states to
their primed or epiblast-like states. This reveals a novel aspect of
SMARCA4 regulation in mammals, organisms in which the regulation of
the promoter proximal pausing of RNAPII has a critically important
To assess the role of chromatin remodelers in controlling RNAPII
pausing, the laboratory used PRO-seq (precision run-on sequencing), a
method for mapping actively transcribing RNAPII with base-pair
resolution. PRO-seq analysis was followed by uncovering changes in DNA
accessibility due to the opening of chromatin with ATAC-seq (assay for
transposase-accessible chromatin using sequencing), and identifying
changes in histone modifications and RNAPII phosphorylation patterns
within the context of chromatin by ChIP-seq (chromatin
immunoprecipitation followed by sequencing). The lab was surprised to
discover that changes in promoter proximal RNAPII pausing are tied to
enhancers and long-range chromatin interactions as revealed by Hi-ChIP
(Hi-C combined with ChIP) and PLAC-seq (proximity ligation assisted
ChIP-seq). These studies provide key insights into the role of
SWI/SNF in enhancer function, long-range chromatin interactions, and
the regulation of gene expression.
INO80 regulation of chromatin dynamics and composition
Deletion of Ino80, the catalytic subunit of the INO80 complex, prevents ventricular compaction in the developing heart and correlates with defective coronary vascularization. Mutations in YY1AP1, another INO80 subunit, disrupt smooth muscle differentiation and are associated with Grange syndrome, an arterial disease. Elevated expression of Ino80 is required for cellular proliferation in several cancers including, melanoma, small cell lung cancer (SCLC), and colon cancer. Further, INO80 is critical for enhancer activity in these same cancers. In melanoma and SCLC, INO80 increases chromatin accessibility at enhancers to promote binding of the Mediator complex by displacing or destabilizing nucleosomes in an poorly understood process.
The Bartholomew group is defining the molecular basis for how INO80 displaces/destabilizes nucleosomes and has found that it is likely distinct from that of the SWI/SNF and SWR1 remodelers. They were the first to show that the motor domain of Ino80 engages nucleosomes near the DNA entry point, 5-6 helical turns from the DNA midpoint, super helical location (SHL) -5/-6. This finding was later confirmed by cryo-EM, and contrasts sharply with the engagement of the motor domains of SWI/SNF and SWR1 with nucleosomes at SHL-2/-3. Additionally, INO80 persistently displaces DNA from the surface of the H2A.Z-H2B dimer closest to its motor domain, leading to H2A.Z exchange, which may provide further clues to the distinct activity of INO80 relative to other remodelers. Further, the lab has shown that INO80 has a strong DNA sequence binding preference and that the specific sequence where the ATPase domain of the Ino80 subunit binds is a key factor influencing the efficiency of the INO80 complex in mobilizing nucleosomes. The Bartholomew group is defining the molecular basis guiding this DNA sequence preference.
INO80 also has nucleosome spacing activity. INO80, by itself, can properly establish the nucleosome-free region that flanks the promoters of many genes. The ability of INO80 to move nucleosomes is highly dependent on linker DNA length. The Bartholomew lab has found that within the INO80 complex, the disengagement of the Arp8 subunit from linker DNA is critical for preventing nucleosomes from moving too close together during remodeling. The binding of Arp8 to linker DNA regulates the interactions of the Arp5 subunit with the histone octamer. When the INO80 complex nears an adjacent nucleosome, the Arp8 module releases from linker DNA leading to the release of Arp5 from the histone octamer. When the Arp subunits are disengaged, the ATPase domain can continue to hydrolyze ATP but ATP hydrolysis no longer drives nucleosome movement.
The unusual dinucleosome specificity of ISW1a is required for regulating early transcription events
In addition to the SWI/SNF and INO80 families, ISWI represents a third remodeler family. ISWI is involved in DNA repair, DNA replication, and organization of higher order chromatin structure. Chromatin structure and ATP-dependent chromatin remodelers (e.g. ISW1a and 1b in yeast) can regulate RNA polymerase II initiation and elongation, as well as cryptic transcription. SMARCA5/SNF2H, one of two ISWI family catalytic subunits in mammals, is overexpressed in both acute myeloid leukemia and aggressive solid tumors.
In yeast, the ISW1a and ISW1b complexes share the same catalytic subunit, Isw1, but have different accessory subunits that confer distinct nucleosome binding and remodeling properties. The Ioc3 subunit of ISW1a uniquely adapts this complex for binding and remodeling dinucleosomes, and inhibits binding and remodeling of mono-nucleosomes. In contrast, ISW1b does not have a preference for dinucleosomes and unlike ISW1a, does not have a linker DNA requirement for remodeling.
In ISW1a, the HLB (helical-linker-DNA-binding) domain of Ioc3 allosterically regulates nucleosome binding and remodeling, which accounts for the nucleosome spacing activity of ISW1a. In vivo, the HLB domain is also required for both the recruitment of ISW1a to chromatin and its dinucleosome binding preference. ISW1a interacts genetically with Yaf9, a protein that is also present in the NuA4 histone acetyltransferase and Swr1 (INO80-related) chromatin remodeling complexes.