5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identified disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/β-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.

Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding RNAs (lncRNAs) have been implicated in tumor drug resistance; however, their potential significance in DDR is still largely unknown. Here, we report that a human lncRNA, CTD-2256P15.2, encodes a micropeptide, named PAR-amplifying and CtIP-maintaining micropeptide (PACMP), with a dual function to maintain CtIP abundance and promote poly(ADP-ribosyl)ation. PACMP not only prevents CtIP from ubiquitination through inhibiting the CtIP-KLHL15 association but also directly binds DNA damage-induced poly(ADP-ribose) chains to enhance PARP1-dependent poly(ADP-ribosyl)ation. Targeting PACMP alone inhibits tumor growth by causing a synthetic lethal interaction between CtIP and PARP inhibitions and confers sensitivity to PARP/ATR/CDK4/6 inhibitors, ionizing radiation, epirubicin, and camptothecin. Our findings reveal that a lncRNA-derived micropeptide regulates cancer progression and drug resistance by modulating DDR, whose inhibition could be employed to augment the existing anticancer therapeutic strategies.

As a deubiquitinating enzyme (DUB), the physiological substrates of ataxin-3 (ATX-3) remain elusive, which limits our understanding of its normal cellular function and that of pathogenic mechanism of spinocerebellar ataxia type 3 (SCA3). Here, we identify p53 to be a novel substrate of ATX-3. ATX-3 binds to native and polyubiquitinated p53 and deubiquitinates and stabilizes p53 by repressing its degradation through the ubiquitin (Ub)-proteasome pathway. ATX-3 deletion destabilizes p53, resulting in deficiency of p53 activity and functions, whereas ectopic expression of ATX-3 induces selective transcription/expression of p53 target genes and promotes p53-dependent apoptosis in both mammalian cells and the central nervous system of zebrafish. Furthermore, the polyglutamine (polyQ)-expanded ATX-3 retains enhanced interaction and deubiquitination catalytic activity to p53 and causes more severe p53-dependent neurodegeneration in zebrafish brains and in the substantia nigra pars compacta (SNpc) or striatum of a transgenic SCA3 mouse model. Our findings identify a novel molecular link between ATX-3 and p53-mediated cell death and provide an explanation for the direct involvement of p53 in SCA3 disease pathogenesis. Ataxin-3 (ATX-3) is a ubiquitously expressed protein that mutated in a neurodegenerative disease called spinocerebellar ataxia type 3 (SCA3). It contains a polyglutamine (polyQ) tract near its C-terminus, the expansion of which is known to be the causative factor for SCA3. It has been known for a long time that ATX-3 is a deubiquitinating enzyme (DUB). However, the substrates targeted by ATX-3 in the physiological context remain elusive, thus largely limiting our understanding of its cellular function and that of the pathogenic mechanism of SCA3. This study has identified p53 to be a novel substrate of ATX-3, and its function is tightly regulated by ATX-3. PolyQ expansion augments ATX-3’s cellular function in p53 regulation. Due to enhanced interaction to p53 and up-regulation of p53, polyQ-expanded ATX-3 led to an increased p53-dependent neuronal cell death in zebrafish and mouse models, thus providing clear in vivo evidences for the direct involvement of p53 in SCA3 pathology. This study not only establishes a basic function of ATX-3 but also provides an explanation of how the interplays between ATX-3 and p53 contribute to the SCA3 pathogenesis; thus, it is an important contribution for the future development of therapeutic approaches for this currently untreatable neurodegenerative disease.

TMCO1 (transmembrane and coiled-coil domains 1) is an endoplasmic reticulum (ER) transmembrane protein that actively prevents Ca²⁺ stores from overfilling. To characterize its physiological function(s), we generated Tmco1^−/− knockout (KO) mice. In addition to the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, Tmco1^−/− females manifest gradual loss of ovarian follicles, impaired ovarian follicle development, and subfertility with a phenotype analogous to the premature ovarian failure (POF) in women. In line with the role of TMCO1 as a Ca²⁺ load-activated Ca²⁺ channel, we have detected a supernormal Ca²⁺ signaling in Tmco1^−/− granulosa cells (GCs). Interestingly, although spontaneous Ca²⁺ oscillation pattern was altered, ER Ca²⁺ stores of germinal vesicle (GV) stage oocytes and metaphase II (MII) arrested eggs were normal upon Tmco1 ablation. Combined with RNA-sequencing analysis, we also detected increased ER stress-mediated apoptosis and enhanced reactive oxygen species (ROS) level in Tmco1^−/− GCs, indicating the dysfunctions of GCs upon TMCO1 deficiency. Taken together, these results reveal that TMCO1 is essential for ovarian follicle development and female fertility by maintaining ER Ca²⁺ homeostasis of GCs, disruption of which causes ER stress-mediated apoptosis and increased cellular ROS level in GCs and thus leads to impaired ovarian follicle development.

DNA polymerase η (Polη) facilitates translesion DNA synthesis (TLS) across ultraviolet (UV) irradiation- and cisplatin-induced DNA lesions implicated in skin carcinogenesis and chemoresistant phenotype formation, respectively. However, whether post-translational modifications of Polη are involved in these processes remains largely unknown. Here, we reported that human Polη undergoes O-GlcNAcylation at threonine 457 by O-GlcNAc transferase upon DNA damage. Abrogation of this modification results in a reduced level of CRL4^CDT2-dependent Polη polyubiquitination at lysine 462, a delayed p97-dependent removal of Polη from replication forks, and significantly enhanced UV-induced mutagenesis even though Polη focus formation and its efficacy to bypass across cyclobutane pyrimidine dimers after UV irradiation are not affected. Furthermore, the O-GlcNAc-deficient T457A mutation impairs TLS to bypass across cisplatin-induced lesions, causing increased cellular sensitivity to cisplatin. Our findings demonstrate a novel role of Polη O-GlcNAcylation in TLS regulation and genome stability maintenance and establish a new rationale to improve chemotherapeutic treatment.