Molecular and Cellular Biology最新文献

One of the primary reasons behind the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL) is the deregulation of the transcription factor BCL11B. The exon 4 of BCL11B harbors several driver mutations, which abolishes its DNA-binding ability. The high frequency of C > T or G > A conversion in close vicinity of AID (Activation-induced cytidine deaminase)-hotspot motifs in the deregulated gene prompted us to investigate the role of AID in BCL11B mutagenesis. Our results reveal that AID is expressed in T-ALL patient-derived cells, binds to BCL11B fragile region (FR) in exon 4 of T cells in vivo, and generates a signature mutation pattern in this region. The mutation frequency in BCL11B FR could be modulated upon overexpression of the AID gene in the knockout background, further suggesting the involvement of AID in BCL11B mutagenesis. Importantly, various lines of experimentation reveal that BCL11B FR could fold into parallel G-quadruplex, triplex, and hairpin structures, which could act as a replication/transcription block, causing mutagenesis. Thus, our results suggest that AID binds to BCL11B exon 4 due to non-B DNA formation, causing U:G mismatches or replication blocks, which, when repaired erroneously, generates deleterious mutations, resulting in loss of functionality of BCL11B, and thus becomes the cause of T-ALL.

Rab11 family interacting protein 4 (Rab11-FIP4) regulates endocytic trafficking. A possible role for Rab11-FIP4 in the regulation of lysosomal function has been proposed, but its precise function in the regulation of cellular homeostasis is unknown. By mRNA array and protein analysis, we found that Rab11-FIP4 is downregulated in the lysosomal storage disease cystinosis, which is caused by genetic defects in the lysosomal cystine transporter, cystinosin. Rescue of Rab11-FIP4 expression in Ctns^-/- fibroblasts re-established normal autophagosome levels and decreased LC3B-II expression in cystinotic cells. Furthermore, Rab11-FIP4 reconstitution increased the localization of the chaperone-mediated autophagy receptor LAMP2A at the lysosomal membrane. Treatment with genistein, a phytoestrogen that upregulates macroautophagy, or the CMA activator QX77 (CA77) restored Rab11-FIP4 expression levels in cystinotic cells supporting a cross-regulation between two independent autophagic mechanisms, lysosomal function and Rab11-FIP4. Improved cellular homeostasis in cystinotic cells rescued by Rab11-FIP4 expression correlated with decreased endoplasmic reticulum stress, an effect that was potentiated by Rab11 and partially blocked by expression of a dominant negative Rab11. Restoring Rab11-FIP4 expression in cystinotic proximal tubule cells increased the localization of the endocytic receptor megalin at the plasma membrane, suggesting that Rab11-FIP4 reconstitution has the potential to improve cellular homeostasis and function in cystinosis.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells increased double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and nonhomologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. To understand how Hhp1 and Hhp2 promote DNA damage repair, we identified new substrates of these enzymes using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 important for DNA repair. Our data suggest that Hhp1 and Hhp2 facilitate DNA repair by phosphorylating multiple substrates, including Arp8.

Smyd1, a member of the Smyd lysine methyltransferase family, plays an important role in myofibrillogenesis of skeletal and cardiac muscles. Loss of Smyd1b (a Smyd1 ortholog) function in zebrafish results in embryonic death from heart malfunction. smyd1b encodes two isoforms, Smyd1b_tv1 and Smyd1b_tv2, differing by 13 amino acids due to alternative splicing. While smyd1 alternative splicing is evolutionarily conserved, the isoform-specific expression and function of Smyd1b_tv1 and Smyd1b_tv2 remained unknown. Here we analyzed their expression and function in skeletal and cardiac muscles. Our analysis revealed expression of smyd1b_tv1 predominately in cardiac and smyd1b_tv2 in skeletal muscles. Using zebrafish models expressing only one isoform, we demonstrated that Smyd1b_tv1 is essential for cardiomyocyte differentiation and fish viability, whereas Smyd1b_tv2 is dispensable for heart development and fish survival. Cellular and biochemical analyses revealed that Smyd1b_tv1 differs from Smyd1b_tv2 in protein localization and binding with myosin chaperones. While Smyd1b_tv2 diffused in the cytosol of muscle cells, Smyd1b_tv1 was localized to M-lines and essential for sarcomere organization in cardiomyocytes. Co-IP analysis revealed a stronger binding of Smyd1b_tv1 with chaperones and cochaperones compared with Smyd1b_tv2. Collectively, these findings highlight the nonequivalence of Smyd1b isoforms in cardiomyocyte differentiation, emphasizing the critical role of Smyd1b_tv1 in cardiac function.

Androgen receptor inhibitors are commonly used for prostate cancer treatment, but acquired resistance is a significant problem. Codeletion of RB and p53 is common in castration resistant prostate cancers, however they are difficult to target pharmacologically. To comprehensively identify gene loss events that contribute to enzalutamide response, we performed a genome-wide CRISPR knockout screen in LNCaP prostate cancer cells. This revealed novel genes implicated in resistance that are largely unstudied. Gene loss events that confer enzalutamide sensitivity are enriched for GSEA categories related to stem cell and epigenetic regulation. We investigated the myeloid lineage stem cell factor HOXA9 as a candidate gene whose loss promotes sensitivity to enzalutamide. Cancer genomic data reveals that HOXA9 overexpression correlates with poor prognosis and characteristics of advanced prostate cancer. In cell culture, HOXA9 depletion sensitizes cells to enzalutamide, whereas overexpression drives enzalutamide resistance. Combination of the HOXA9 inhibitor DB818 with enzalutamide demonstrates synergy. This demonstrates the utility of our CRISPR screen data in discovering new approaches for treating enzalutamide resistant prostate cancer.