Access Type

Open Access Embargo

Date of Award

January 2021

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary Kay H. Pflum

Abstract

Regulators of chromatin structure have emerged as a key driver of transcriptional responses inside the cell. Two such groups of regulators, histone writers and erasers; the proteins, that add or remove histone post translational modifications (PTMs), have become the central players in chromatin structure. Thus, the aberrant expression of writers and erasures is a hallmark in human diseases. For example, overexpression of the erasures histone deacetylase1 (HDAC1) and lysine specific demethylase1 (LSD1) had been reported in many cancers. Currently, HDAC inhibitors have been used successfully for cancer treatment and several inhibitors targeting LSD1 are in clinical trial. To date, apart from histones, only few non-histone substrates have been identified for both HDAC1 and LSD1, which has delayed disease diagnosis and drug development. Therefore, a systematic tool linking epigenetic erasures to non-epigenetic substrates and corresponding mechanisms of action are an unmet need in the field.

To augment discovery of non-histone substrates, we recently pioneered a simple method called substrate trapping to isolate HDAC1 substrates using an inactive mutant. Our prior work documented that different HDAC1 mutants preferentially bound different substrates, suggesting that multiple mutants should be used for efficient trapping. To test the value of trapping with multiple separate mutants, a proteomics-based study was performed using three optimal HDAC1 mutants (H141A, F150A and C151A) simultaneously. The data suggest that trapping with three separate mutants uniquely identified a number of potential substrates, although trapping with a single mutant remains simple and effective.

The many successes we had with HDAC1 substrate trapping led us to study the generality of the substrate trapping tool by applying to LSD1. In this study, inactive LSD1 mutant, K661A, was used to profile substrates of LSD1. Among the substrate hits, BAG2 and SLFN11 were validated as LSD1 substrates. These newly discovered substrates are involved in various biological processes, suggesting novel functions of LSD1 apart from epigenetics. Our findings reveal for the first time a list of candidate substrates for LSD1 and an efficient approach to profile substrates of lysine demethylases (KDM).

Unlike HDAC1, LSD1 known to have a considerably large active site, which allows binding of at least 21 amino acid residues to the active site. Due to the presence of large active site and based on available crystal structure data on histone tail binding to the active site of LSD1, we hypothesized LSD1 to have some level of substrate specificity. To test this hypothesis, we studied the binding of several known LSD1 substrates to four selective inactive mutants of LSD1. Data clearly showed the presence of two substrate binding orientations with distinct sequence specificities, indicating a level of LSD1 substrate specificity.

In summary, with this work we have uncovered unanticipated HDAC1 and LSD1 biology through the unbiased identification of substrates and mechanisms of action. Given the role of these proteins in epigenetic, and their possible role in human disease, this work has the potential to augment our understanding of cell biology and embolden drug design efforts targeting epigenetic erasures HDAC1 and LSD1.

Available for download on Thursday, January 05, 2023

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Biochemistry Commons

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