Exogenous Control of the Assembly of Transcriptional Complexes at Gene Promoters.

Establishment of gene expression patterns defines cellular function and the first critical step in this process is transcription. Exogenous agents that can alter transcription hold tremendous utility as chemical genetic probes and as therapeutics. In my dissertation research I have pursued strategies for controlling the transcription of genes with small drug-like molecules. The transcriptional status of genes is defined by coregulatory complexes recruited to gene promoters. To achieve control of this recruitment, small molecules that bind to these proteins must be discovered. By use of a high-throughput biochemical screen, sekikaic acid was discovered as a potent ligand of the transcriptional coactivator protein CBP. Sekikaic acid binds to the KIX domain of CBP and its binding precludes the in vitro interaction of transcriptional activation domains from the human transcription factors MLL and CREB to two distinct sites on KIX. In order for a small molecule to recruit target proteins to individual genes, it must have the ability to localize to DNA. Nuclear receptors were identified as potential targets that could tether bifunctional molecules to their target genes. Conjugates of glucocorticoid receptor (GR) ligands and a ligand of the FK506 binding protein (FKBP) were prepared, and it was demonstrated that the conjugates bind the receptor and recruit an FKBP fusion protein that influence the transcriptional control of target genes in concert with intrinsic GR activity. The ability to recruit nuclear receptor extrinsic functionality to target genes with receptor targeting bifunctional molecules has potential to greatly increase our level of control over the therapeutically relevant genes that can be targeted via nuclear receptors. The traditional paradigm of artificial transcription factor design is an attempt to mimic natural transcription factors by binding their direct interaction partners. The arsenal of ligands that bind these proteins is limiting, and instead untraditional recruitment targets have been identified: the enzymatic subunits of chromatin modifying complexes. High affinity ligands of these subunits have been discovered and it is demonstrated in this work that a histone deacetylase inhibitor can be incorporated into NR targeting bifunctional recruiters that retain the ability of each conjugate partner to bind its respective protein target.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89742/1/jonaswh_1.pd

Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division

A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-014-1338-3) contains supplementary material, which is available to authorized users