The long-term goal of our research is to investigate the molecular mechanisms by which epigenetic alterations of chromatin structure promote human developmental disorders and diseases such as Floating- Harbor syndrome (FHS). It was recently found that mutations of the SRCAP (SNF2-related CBP activator protein) gene cause FHS, which is a rare dominant disorder characterized by proportionate short stature with dysmorphic facial features, delayed osseous maturation, and delayed speech development. However, the molecular bases underlying the disease remain to be elucidated. SRCAP is a member of the SNF2 family of ATP-dependent chromatin remodeling enzymes, and forms a 12-subunit, large protein complex. All of SRCAP mutations in FHS patients are heterozygous truncating alleles, tightly clustered within the final 33th and 34th exons, suggesting that the C-terminal domain of SRCAP is crucial for the SRCAP function. Importantly, it was reported that individuals carrying a deletion of a chromosomal region containing the SRCAP gene have no reported phenotype, suggesting that SRCAP deletion is haplosufficient. The SRCAP complex is required for the incorporation of histone variant H2A.Z into nucleosomes. H2A.Z is deposited within promoter-proximal nucleosomes, and plays essential roles in transcription, genome stability, and proper stem cell differentiation. The overall objective of this proposed research is to characterize the SRCAP complex, and determine how SRCAP mutations alter epigenetic chromatin structure and function, thus resulting in FHS. Our overall strategy in this proposal is to exploit a powerful combination of biochemical, biophysical, and genomics techniques to dissect the molecular mechanisms by which the SRCAP complex regulates H2A.Z deposition and the truncated SRCAP causes FHS.
In Aim 1, we will dissect the mechanism of H2A.Z deposition by the SRCAP complex. The molecular mechanism by which the SRCAP complex catalyzes H2A.Z deposition is largely unknown, mainly due to the limited protein availability, as SRCAP forms a large multi-protein complex. To address this, we have successfully reconstituted the whole SRCAP complex from individual, recombinant subunits using the Multibac baculovirus expression system. We will define the detailed biochemical properties, especially histone exchange activity, of the SRCAP complex. We will employ various chromatin remodeling assays including FRET-based assays. Furthermore, we will investigate the function of SRCAP in mouse embryonic stem cells (ESCs), since H2A.Z is necessary for ESC differentiation. We will perform genomics analyses to dissect how SRCAP regulates the epigenetic landscape of H2A.Z during ESC differentiation.
In Aim 2, we will define the effects of Floating-Harbor syndrome mutations on the SRCAP complex. Although all SRCAP mutations in FHS patients are heterozygous truncating alleles, it remains unclear how the truncated SRCAP produces a dominant negative effect. To define the effects of FHS mutations on the SRCAP function, we will reconstitute the SRCAP complex containing a FHS mutation, and dissect how it affects the assembly and function of the SRCAP complex. Then, we will test two hypotheses for the dominant negative effect of the truncated SRCAP by employing various biophysical and genomics approaches. !

Public Health Relevance

Floating-Harbor syndrome (FHS) is a rare dominant developmental disorder characterized by proportionate short stature with dysmorphic facial features, delayed osseous maturation, and delayed speech development. This proposed research will provide mechanistic understanding of how SRCAP mutations cause FHS, and could lead to new therapeutic strategy. !

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
1R03HD095088-01A1
Application #
9746250
Study Section
National Institute of Child Health and Human Development Initial Review Group (CHHD)
Program Officer
Parisi, Melissa
Project Start
2019-07-11
Project End
2021-06-30
Budget Start
2019-07-11
Budget End
2020-06-30
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655