LSD1 is the first described histone demethylase which demethylates H3K4me1/2 (Shi et el., 2004), thus, causing transcriptional repression. Alternatively, LSD1 was demonstrated to have H3K9me1/2 demethylase activity when bound by androgen receptor, hence, causing transcriptional activation (Schule et al., 2005). LSD1 is commonly recruited by the so called CoREST core complex including: RCOR1, HDAC1 and HDAC2 among others and therefore is coupled with histone deacetylation and transcriptional repression (Foster et al., 2010). It is an important regulator of pluripotency in early development and it occupies, along with pluripotency factors NANOG and OCT4, the promoters of major lineage determining genes that are poised for activation in the pluripotent state, (Adamo et al., 2011). There are four described isoforms for LSD1: LSD1, LSD1-E2a, LSD1-8a and LSD1-E2a/E8a (Zibetti et al., 2010). While the Cterminus of LSD1 is extensively studied and the function of the isoforms LSD1-E8a and LSD1-E8aE2a is described, there is scarce knowledge on LSD1 N-terminus unstructured region and the SWIRM domain. In this project I examined the role of the differently spliced exon 2a on the function of the SWIRM domain through generation of eight constructs coding for the N-terminal portion of LSD1 SV1 and SV2 fused with a C- or N-terminus FLAG tag. I then performed an immunoprecipitation experiment followed by mass spectrometry and proteomics analysis that led to the identification of previously unknown binding partners to the LSD1 SWIRM domain: NONO and IGF2B3.

<p>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.</p><p>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.</p><p>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.</p><p>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</p><p>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.</p> / Dissertation

Oral squamous cell carcinoma (OSCC), or oral cancer, accounts for the majority of head and neck cancers. Resistance to therapy is a challenge, and 5-year survival rate remains at ~50 percent. Lysine-specific demethylase 1 (LSD1) plays a crucial role in controlling cell homeostasis in health and disease. LSD1 is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. LSD1 is a nuclear histone demethylase that has been implicated in maintaining the undifferentiated state of cancer-initiating stem cells and promoting OSCC. Large dataset analysis showed that genetic alterations, including upregulation of LSD1, are seen in clinical cancers including OSCC. This study aims to evaluate the unknown mechanism of LSD1 and determine if pharmacologic inhibition of LSD1 has preventative and/or therapeutic applications for OSCC. This study used the 4NQO mouse model to induce OSCC in mice and split the mice into 8 treatment groups. Each group received a different immunotherapy treatment (SP2509, Verteporfin, anti PD-1 and anti PD-L1 alone and in combination). Our results have shown that LSD1 inhibition reduces the development of gross pathologic lesions. LSD1 inhibition has also shown to cause differences in gene expression in preneoplasia and OSCC, attenuating many genes that are part of the pro-oncogenic gene network (LSD1, YAP, EGFR), immune checkpoints (PD-1 and PD-L1), and Hippo signaling effectors (YAP, TAZ). Interestingly, LSD1 has shown a role in regulating the immune microenvironment and promoting antitumor immunity, which led us to investigate LSD1 in combination with immune checkpoint antibodies (anti PD-1 and anti PD-L1). Our results show that LSD1 sensitizes to anti-PD-1 and anti-PD-L1 antibodies to treat mouse tongue OSCC. Thus, we showed for the first time that blocking LSD1 inhibits preneoplasia and OSCC feed-forward loop, which could have implications in OSCC prevention, chemo- and immunotherapeutic combinations.

The epigenetic mechanisms that connect hormone signaling to chromatin remain largely unknown. Here we show that LSD1/KDM1A is a critical glucocorticoid receptor (GR) coactivator and report a previously unexplored mechanism where LSD1 activates gene transcription through H3K4me2 demethylation. We demonstrate that direct interaction of GR with LSD1 primarily inhibit its activity against H3K4me1 in vitro. While this interaction enables GR to recruit LSD1 in vivo and allows loss of H3K4me2, it impedes further demethylation. Thus resulting in conversion of H3K4me2 to H3K4me1 at enhancers and promotes H3K27 acetylation and gene activation. We also find that H3K4me2 is an early enhancer mark predicting GR and LSD1 recruitment. These findings differ from the reported mechanism for ER and AR-mediated gene activation, providing a novel mechanism for LSD1 coactivator function as well as shed light on the role of H3K4me2 and enhancers in hormone-mediated gene regulation. In addition we present evidence supporting never before characterized H3K79me3 demethylase activity by members of the JMJD2 family of proteins.