CW-type Zinc Finger Research Articles

Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

Comparative Study of Autoantibody Responses between Lung Adenocarcinoma and Benign Pulmonary Nodules

IntroductionThe reduction in lung cancer mortality associated with computed tomography (CT) screening has led to its increased use and a concomitant increase in the detection of benign pulmonary nodules. Many individuals found to have benign nodules undergo unnecessary, costly, and invasive procedures. Therefore, there is a need for companion diagnostics that stratify individuals with pulmonary nodules into high-risk or low-risk groups. Lung cancers can trigger host immune responses and elicit antibodies against tumor antigens. The identification of these autoantibodies (AAbs) and their corresponding antigens may expand our knowledge of cancer immunity, leading to early diagnosis or even benefiting immunotherapy. Previous studies were performed mostly in the context of comparing cancers and healthy (smoker) controls. We have performed one of the first studies to understand humoral immune response in patients with cancer, patients with benign nodules, and healthy smokers. MethodsWe first profiled seroreactivity to 10,000 full-length human proteins in 40 patients with early-stage lung cancer and 40 smoker controls by using nucleic acid programmable protein arrays to identify candidate cancer-specific AAbs. Enzyme-linked immunosorbent assays of promising candidates were performed on 137 patients with lung cancer and 127 smoker controls, as well as on 170 subjects with benign pulmonary nodules. ResultsFrom protein microarray screening experiments using a discovery set of 40 patients and 40 smoker controls, 17 antigens showing higher reactivity in lung cancer cases relative to the controls were subsequently selected for evaluation in a large sample set (n = 264) by using enzyme-linked immunosorbent assay. A five-AAb classifier (tetratricopeptide repeat domain 14 [TTC14], B-Raf proto-oncogene, serine/threonine kinase [BRAF], actin like 6B [ACTL6B], MORC family CW-type zinc finger 2 [MORC2], and cancer/testis antigen 1B [CTAG1B]) that can differentiate lung cancers from smoker controls with a sensitivity of 30% at 89% specificity was developed. We further tested AAb responses in subjects with CT-positive benign nodules (n = 170), and developed a five-AAb panel (keratin 8, type II, TTC14, Kruppel-like factor 8, BRAF, and tousled like kinase 1) with a sensitivity of 30% at 88% specificity. Interestingly, messenger RNA levels of six AAb targets (TTC14, BRAF, MORC family CW-type zinc finger 2, cancer/testis antigen 1B, keratin 8, type II, and tousled like kinase 1) were also found to increase in lung adenocarcinoma tissues based on The Cancer Genome Atlas data set. ConclusionWe discovered AAbs associated with lung adenocaricnoma that have the potential to differentiate cancer from CT-positive benign diseases. We believe that these antibodies warrant future validation using a larger sample set and/or longitudinal samples individually or as a panel. They could potentially be part of companion molecular diagnostic modalities that will benefit subjects undergoing CT screening for lung cancer.

Mutations in the MORC2 gene cause axonal Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is a complex disorder with wide genetic heterogeneity. Here we present a new axonal Charcot-Marie-Tooth disease form, associated with the gene microrchidia family CW-type zinc finger 2 (MORC2). Whole-exome sequencing in a family with autosomal dominant segregation identified the novel MORC2 p.R190W change in four patients. Further mutational screening in our axonal Charcot-Marie-Tooth disease clinical series detected two additional sporadic cases, one patient who also carried the same MORC2 p.R190W mutation and another patient that harboured a MORC2 p.S25L mutation. Genetic and in silico studies strongly supported the pathogenicity of these sequence variants. The phenotype was variable and included patients with congenital or infantile onset, as well as others whose symptoms started in the second decade. The patients with early onset developed a spinal muscular atrophy-like picture, whereas in the later onset cases, the initial symptoms were cramps, distal weakness and sensory impairment. Weakness and atrophy progressed in a random and asymmetric fashion and involved limb girdle muscles, leading to a severe incapacity in adulthood. Sensory loss was always prominent and proportional to disease severity. Electrophysiological studies were consistent with an asymmetric axonal motor and sensory neuropathy, while fasciculations and myokymia were recorded rather frequently by needle electromyography. Sural nerve biopsy revealed pronounced multifocal depletion of myelinated fibres with some regenerative clusters and occasional small onion bulbs. Morc2 is expressed in both axons and Schwann cells of mouse peripheral nerve. Different roles in biological processes have been described for MORC2. As the silencing of Charcot-Marie-Tooth disease genes have been associated with DNA damage response, it is tempting to speculate that a deregulation of this pathway may be linked to the axonal degeneration observed in MORC2 neuropathy, thus adding a new pathogenic mechanism to the long list of causes of Charcot-Marie-Tooth disease.

By recruiting HDAC1, MORC2 suppresses p21 Waf1/Cip1 in gastric cancer.

Microrchidia (MORC) family CW-type zinc-finger 2 (MORC2) regulates chromatin remodeling during the DNA-damage response, represses gene transcription, promotes lipogenesis. Here, we found that MORC2 down-regulated p21 by recruiting HDAC1 to the p21 promoter, in a p53-independent manner. MORC2-mediated down-regulation of p21 in turn promoted cell cycle progression in gastric cancer cells. Furthermore, MORC2 expression correlated negatively with p21 expression in gastric tumors in patients. We suggest that MORC2 may be a potential therapeutic target in cancer.

PAK1-mediated MORC2 phosphorylation promotes gastric tumorigenesis.

To date, microrchidia (MORC) family CW-type zinc-finger 2 (MORC2), has been found to be involved in p21-activated kinase1 (PAK1) pathway to maintain genomic integrity. Here, we explore its novel role in cancer. We demonstrate that PAK1-mediated MORC2 phosphorylation promotes cell cycle progression, defective phosphorylation of MORC2-S677A results in attenuated cell proliferation and tumorigenicity of gastric cancer cells, which is significantly enhanced in overexpression of phospho-mimic MORC2-S677E form, suggesting the importance of MORC2 phosphorylation in tumorigenesis. More importantly, phosphorylation of MORC2 correlates positively with PAK1 expression in clinical gastric cancer. Furthermore, high expression of PAK1 and phosphorylation of MORC2 appear to be associated with poor prognosis of clinical gastric cancer. Collectively, these findings revealed a novel function of MORC2 phosphorylation in promoting gastric cell proliferation in vitro and tumorigenesis in vivo, suggesting that blocking PAK1-mediated MORC2 phosphorylation might be a potential therapeutic strategy for gastric tumorigenesis.

MORC1 exhibits cross-species differential methylation in association with early life stress as well as genome-wide association with MDD.

Early life stress (ELS) is associated with increased vulnerability for diseases in later life, including psychiatric disorders. Animal models and human studies suggest that this effect is mediated by epigenetic mechanisms. In humans, epigenetic studies to investigate the influence of ELS on psychiatric phenotypes are limited by the inaccessibility of living brain tissue. Due to the tissue-specific nature of epigenetic signatures, it is impossible to determine whether ELS induced epigenetic changes in accessible peripheral cells, for example, blood lymphocytes, reflect epigenetic changes in the brain. To overcome these limitations, we applied a cross-species approach involving: (i) the analysis of CD34+ cells from human cord blood; (ii) the examination of blood-derived CD3+ T cells of newborn and adolescent nonhuman primates (Macaca mulatta); and (iii) the investigation of the prefrontal cortex of adult rats. Several regions in MORC1 (MORC family CW-type zinc finger 1; previously known as: microrchidia (mouse) homolog) were differentially methylated in response to ELS in CD34+ cells and CD3+ T cells derived from the blood of human and monkey neonates, as well as in CD3+ T cells derived from the blood of adolescent monkeys and in the prefrontal cortex of adult rats. MORC1 is thus the first identified epigenetic marker of ELS to be present in blood cell progenitors at birth and in the brain in adulthood. Interestingly, a gene-set-based analysis of data from a genome-wide association study of major depressive disorder (MDD) revealed an association of MORC1 with MDD.

Cytosolic functions of MORC2 in lipogenesis and adipogenesis

Microrchidia (MORC) family CW-type zinc finger 2 (MORC2) has been shown to be involved in several nuclear processes, including transcription modulation and DNA damage repair. However, its cytosolic function remains largely unknown. Here, we report an interaction between MORC2 and adenosine triphosphate (ATP)-citrate lyase (ACLY), an enzyme that catalyzes the formation of acetyl-coA and plays a central role in lipogenesis, cholesterogenesis, and histone acetylation. Furthermore, we demonstrate that MORC2 promotes ACLY activation in the cytosol of lipogenic breast cancer cells and plays an essential role in lipogenesis, adipogenesis and differentiation of 3T3-L1 preadipocytic cells. Consistently, the expression of MORC2 is induced during the process of 3T3-L1 adipogenic differentiation and mouse mammary gland development at a stage of increased lipogenesis. This observation was accompanied by a high ACLY activity. Together, these results demonstrate a cytosolic function of MORC2 in lipogenesis, adipogenic differentiation, and lipid homeostasis by regulating the activity of ACLY.

Structure-function analysis reveals a novel mechanism for regulation of histone demethylase LSD2/AOF1/KDM1b.

LSD2/AOF1/KDM1b catalyzes demethylation of mono- and di-methylated H3K4 and plays an important role in transcriptional regulation and genomic imprinting. Here, we report the high-resolution crystal structures of apo-LSD2 and LSD2 in complex with a peptide that mimics H3K4me2. Three structural domains of LSD2, namely, the novel N-terminal zinc finger, the centrally located SWIRM domain, and the C-terminal oxidase domain, closely pack together to form a boot-shaped structure. The active site cavity in the oxidase domain is large enough to accommodate several residues of the histone H3 tail and cannot discriminate between the different states of H3K4 methylation. The N-terminal zinc-finger domain, composed of a novel C4H2C2-type zinc finger and a specific CW-type zinc finger, is required for demethylase activity and, surprisingly, the binding of cofactor flavin adenine dinucleotide (FAD). In fact, a relay of extensive interactions through the zinc finger-SWIRM-oxidase domains is required for LSD2 demethylase activity and the binding of FAD. These results reveal a novel mechanism for the zinc finger and SWIRM domains in controlling LSD2 demethylase activity and provide a framework for elucidating the regulation and function of LSD2.

MORC2 Signaling Integrates Phosphorylation-Dependent, ATPase-Coupled Chromatin Remodeling during the DNA Damage Response

Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.

Two-step colocalization of MORC3 with PML nuclear bodies

Many functional subdomains, including promyelocytic leukemia nuclear bodies (PML NBs), are formed in the mammalian nucleus. Various proteins are constitutively or transiently accumulated in PML NBs in a PML-dependent manner. MORC3 (microrchidia family CW-type zinc-finger 3), also known as NXP2, which consists of GHL-ATPase, a CW-type zinc-finger and coiled-coil domains, is localized in PML NBs, where it recruits and activates p53 to induce cellular senescence. Interestingly, we found that MORC3 can form PML-independent nuclear domains (NDs) in mouse hematopoietic cells and even in Pml-deficient cells. Here, we show that MORC3 colocalizes with PML by a two-step molecular mechanism: the PML-independent formation of MORC3 NDs by the ATPase cycle, and the association of MORC3 with PML via the SUMO1-SUMO-interacting motif (SIM). Similarly to other members of the GHL-ATPase family, MORC3 functions as a 'molecular clamp'. ATP binding induces conformational changes in MORC3, leading to the formation of MORC3 NDs, and subsequent ATP hydrolysis mediates the diffusion and binding of MORC3 to the nuclear matrix. MORC3 might clamp DNA or nucleosomes in MORC3 NDs via the CW domain. Furthermore, the SUMOylation of MORC3 at five sites was involved in the association of MORC3 with PML, and SUMO1-unmodified MORC3 formed NDs independently of PML.

Involvement of histone deacetylation in MORC2-mediated down-regulation of carbonic anhydrase IX

Carbonic anhydrase IX (CAIX) plays an important role in the growth and survival of tumor cells. MORC2 is a member of the MORC protein family. The MORC proteins contain a CW-type zinc finger domain and are predicted to have the function of regulating transcription, but no MORC2 target genes have been identified. Here we performed a DNA microarray hybridization and found CAIX mRNA to be down-regulated 8-fold when MORC2 was overexpressed. This result was further confirmed by northern and western blot analysis. Our results also showed that the protected region 4 (PR4) was important for the repression function of MORC2. Moreover, MORC2 decreased the acetylation level of histone H3 at the CAIX promoter. Meanwhile, trichostatin A (TSA) had an increasing effect on CAIX promoter activity. Among the six HDACs tested, histone deacetylase 4 (HDAC4) had a much more prominent effect on CAIX repression. ChIP and ChIP Re-IP assays showed that MORC2 and HDAC4 were assembled on the same region of the CAIX promoter. Importantly, we further confirmed that both proteins are simultaneously present in the PR4-binding complex. These results may contribute to understanding the molecular mechanisms of CAIX regulation.

A Novel Mammalian Flavin-dependent Histone Demethylase

Methylation of Lys residues on histone proteins is a well known and extensively characterized epigenetic mark. The recent discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled. Among the histone demethylases so far identified, LSD1 has the unique feature of functioning through a flavin-dependent amine oxidation reaction. Data base analysis reveals that mammalian genomes contain a gene (AOF1, for amine-oxidase flavin-containing domain 1) that is homologous to the LSD1-coding gene. Here, we demonstrate that the protein encoded by AOF1 represents a second mammalian flavin-dependent histone demethylase, named LSD2. The new demethylase is strictly specific for mono- and dimethylated Lys4 of histone H3, recognizes a long stretch of the H3 N-terminal tail, senses the presence of additional epigenetic marks on the histone substrate, and is covalently inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a biochemically stable complex with the C-terminal domain of the corepressor protein CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites that are not present in LSD1. We conclude that mammalian LSD2 represents a new flavin-dependent H3-Lys4 demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of chromatin-remodeling complexes that are distinct from those involving LSD1.