Browsing by Subject "KDM1A"

The posttranslation modification of histone proteins within the nucleosomes of chromatin plays important roles in the regulation of gene expression in both normal biological and pathobiological processes. These modifications alter local chromatin structure and subsequently alter the expression profile of associated genes. Histone methylation, which was long thought immutable, is one such modification that plays a dual functionality in both activation and repression of gene expression and can be thought of as an information storage mark. With the initial discovery of lysine-specific demethylase 1A (LSD1/KDM1A), an FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively, the missing counterbalance to dynamic histone methylation was cemented. This discovery further strengthened the link between histone demethylation and transcriptional regulation and the enzyme has since been identified as a target with therapeutic potential.

Given the significance of KDM1A enzymatic activity, herein we report our efforts to characterize novel binding interactions that dictate the enzymes biological and pathobiological functions. As KDM1A falls into the greater class of flavin-dependent amine oxidases, it contains features that are recurrent within the class, but due to its unique ability to work on histone and non-histone substrates has unprecedented structural elements. Although the active site is expanded compared to the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint of the histone H3 tail. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. Using steady-state kinetic analyses, we now show that unmodified histone H3 is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM that is approximately 100-fold higher than the 21-mer peptide product. The relative affinity of dose-response curves is independent of preincubation time suggesting that H3 rapidly reaches equilibrium with KDM1A. Rapid dilution experiments confirmed the increased binding affinity of full-length H3 toward KDM1A was at least partially caused by a slow off-rate with a koff of 0.072 min-1, a half-life (t1/2) of 9.63 min, and residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction revealing a Kd of 9.02 ± 2.27 nM, a kon of 9.26 x 104 ± 1.5 x 104 M-1s-1 and koff of 8.35 x 10-4 ± 3.4 x 105 s-1. Additionally, consistent with H3 being the only histone substrate of KDM1A, no other core histones are inhibitors of demethylation activity. Our data suggests that KDM1A contains a histone H3 secondary specificity recognition element on the enzyme surface and required further characterization.

In order to characterize this secondary H3 binding site, we turned to the use of cysteine labeling, chemical cross-linking coupled to proteolysis and LC-MS/MS, HDX-MS, and the design of an active, tower domain deletion KDM1A mutant. We now show that the tower domain contributes to the extended binding interface of the KDM1A/H3 interaction. Additionally, we show that the KDM1A tower domain is not required for demethylation activity and that one can functionally uncouple catalytic activity from protein-protein interactions that occur along the KDM1A tower domain interface, a domain unprecedented in the greater amine oxidase family. Furthermore, this towerless mutant will be useful for dissecting molecular contributions to KDM1A function along the tower domain. Our discovery of this secondary binding site within the aforementioned domain points to how pivotal this region is to the control and localization of KDM1A enzymatic activity as it also serves a pivotal role as a protein-protein interaction motif for the nucleation of a multitude of multimeric protein complexes.

With this in mind, we set out to design a strategy to isolate the core histone demethylase complex from E. coli cellular lysates. With the use of polycistronic vectors that encode both KDM1A and CoREST for coexpression we were able to produce appreciable amounts of chromatographically pure complex. As our CoREST construct in this strategy contains both the ELM2 and SANT2 domain needed for interaction with the HDACs, this core complex will serve as a starting point for future work that will tease apart additional influences on substrate binding and recognition imparted on KDM1A from binding partners. This preparation can therefore be used in a multitude of downstream studies including reconstitution of the core histone demethylase/deactylase complex and in depth kinetic and biophysical analyses and provides an invaluable starting point

This work provides a foundational understanding of this unprecedented secondary binding site on the surface of the KDM1A tower domain and how it may play an important role in substrate and product recognition. We suspect that this extended interaction interface may control KDM1A localization within specific chromatin loci and allow the enzyme to serve as a docking element for the nucleation of protein complexes or transcriptional machinery. On the other hand, disruption of this point of contact between the KDM1A/H3 binary complex may also facilitate enzyme/product dissociation, thereby tuning the catalytic activity of the demethylase. Additionally, the ability to produce substantial quantities of the core histone demethylase complex is a necessary step in the decoding of the ‘histone code’ hypothesis of KDM1A and its associated complexes. We suspect that the body of this work will prove to be invaluable for future characterization of the enzyme and its role in biology and pathobiology.

The post translational modification (PTM) of histone proteins is a highly dynamic process that is utilized in the control of gene transcription. This epigenetic process involves enzymatic ‘writers’ and ‘erasers’ which place or remove chemical modifications to the unstructured tails of histone proteins which protrude out from the nucleosomal core. In a highly dynamic manner, each PTM is spatiotemporally regulated and combinations of PTMs at a gene promotor or enhancer region leads to transcriptional enhancement or repression. The gene targets as well as selectivity and specificity of epigenetic enzymes is regulated by the multimeric complexes each enzyme is co-opted. Each complex contains a unique set of coregulatory proteins with RNA and DNA binding domains and PTM ‘reader’ domains to direct the catalytic machinery to a specific subset of genes. The coregulatory proteins also affect the specificity and selectivity of the enzyme through mechanisms which are only beginning to be explored.

Our interest is in elucidating the role of coregulatory proteins and lncRNA with respect to lysine-specific demethylase 1 (LSD1/KDM1A). A flavin-dependent mono-and di-demethylase of H3K4me1/2 and H3K9me1/2, KDM1A has been implicated in many different multimeric enzymatic complexes which, in some cases such as the REST and NuRD complexes, function on opposing pathways. This disparity in the downstream outcome being coordinated by the same enzyme highlights the need to understand not only epigenetic enzymes, but to consider the complexes as a whole towards therapeutic targeting.

The specific aims of my thesis were to (a) interrogate the role of individual and multiple coregulatory partners in enzyme selectivity and specificity (b) establish tools to study the mechanisms of biochemical and biophysical of protein:protein and protein:lncRNA interactions and (c) elucidate key characteristics of protein:protein and protein:lncRNA interfaces towards targeted disruption. To this end, I have utilized cloning and mutagenesis methods to heterologously express and purify coregulatory partners of KDM1A in E. coli. I chose coregulatory partners found in a common catalytic core as well as several additional coregulatory proteins from a stable KDM1A-containing 5-mer complex. I have produced multiple constructs for four of these proteins to allow for multiple affinity purification routes as well as for future binding studies. I have further expressed each of these constructs and have made significant efforts towards the purification of each construct based on solubility.

I furthermore established HDX-MS and SELEX protocol in our lab as tools to allow us to explore the dynamics of these epigenetic interactions. I further demonstrated and confirmed that there is no hotspot along the binding interface between KDM1A and CoREST, but that CoREST stabilizes the apical end of the KDM1A tower domain via HDX-MS with the highest change in deuterium uptake, over 20%, long KDM1A TαA residues 440-451.

I also made significant efforts towards elucidating the interaction between KDM1A and HOTAIR. Firstly, I established an RNA radiolabeled EMSA assay for the lab which allowed us to test the binding of HOTAIR to KDM1A. With this assay, we saw that CoREST286-482, specifically the linker region (residues 293-380), must be bound to KDM1A for HOTAIR to bind and that the dissociation constant was unchanged at 1.710.38 µM and 1.29±0.34 µM, respectively. Further, I confirmed that the first 320 nt of domain 4 of HOTAIR (nt 1500-1820) contain the critical binding and that the dissociation constant was slightly higher at 2.97±0.96 µM.

I have also optimized SHAPE-MaP and crosslinking strategies to explore the binding interface between KDM1A:CoREST286-482 and lncRNA. I determined that there were 83 nt that displayed at least a 1.5-fold change in SHAPE reactivity of HOTAIR D4 due to the presence of KDM1A:CoREST286-482. I also utilized a free-energy based secondary structure model to establish a secondary structure for HOTAIR D4 based on my SHAPE-MaP data. I noted that 44% of the significant nt were confined to a stretch of RNA (nt 1538-1610, 1779-1844) that is predominantly dsRNA. Further usage of photochemical crosslinking strategies revealed a propensity for G:C paired nt to be crosslinked to KDM1A:CoREST286-482. A similar nt sequence around these paired nt suggests a binding motif. The implications of these results is discussed herein.

Lysine-specific demethylase 1 (LSD1/KDM1A) regulates transcriptional events by post-translational modifications of histone H3 tails at residues K4 an K9. This enzyme plays a vast number of roles in both normal cellular functions and diseases states. Increasingly it is appreciated that this enzyme, like most epigenetic regulators, does not function alone, but rather forms a catalytic subunit of much larger protein assemblies that congregate on chromatin to concertedly mediate transcriptional events. LSD1 in particular has been found in many different complexes, in many different tissues and can facilitate both activation and repression events.

Because of these roles, LSD1 is viewed as a potential therapeutic target. Significant effort has recently led to the development of highly selective and potent active-site inhibitors. These inhibitors have particularly shed light on the cancer-promoting activities of LSD1 in acute myeloid leukemia and small cell lung carcinoma. However, one failing of these strategies is that active site inhibition is incapable of differentiating between the multitude of functions LSD1 performs. We sought to address this issue by instead developing first-generation tools to explore protein-protein interaction disruption as an alternative strategy for inhibiting the enzyme.

To this end, we have carefully examined a well-characterized interaction between LSD1 and the scaffolding protein CoREST. Using this interaction as a template, we developed a probe we show can compete with CoREST for interaction with LSD1. Furthermore, we generated cell permeable versions of this probe and examined the effects in a model of breast cancer. We find that our probe can selectively inhibit estrogen signaling, a feat that was not possible with current small molecule inhibition or RNA interference technologies. We therefore we propose that disrupting interactions such as this is an excellent alternative for targeting “undruggable” proteins, but also may also expand the current therapeutic space by granting precise control over the individual functions of proteins.