Role of protein kinase PLK1 in the epigenetic maintenance of centromeres.

The centromere, a chromosome locus defined by the histone H3-like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.

PLK1-mediated phosphorylation cascade activates Mis18 complex to ensure centromere inheritance.

Accurate chromosome segregation requires the attachment of microtubules to centromeres, epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, CENP-A nucleosomes undergo dilution. To preserve centromere identity, correct amounts of CENP-A must be restored in a cell cycle-controlled manner orchestrated by the Mis18 complex (Mis18α-Mis18ß-Mis18BP1). We demonstrate here that PLK1 interacts with the Mis18 complex by recognizing self-primed phosphorylations of Mis18α (Ser54) and Mis18BP1 (Thr78 and Ser93) through its Polo-box domain. Disrupting these phosphorylations perturbed both centromere recruitment of the CENP-A chaperone HJURP and new CENP-A loading. Biochemical and functional analyses showed that phosphorylation of Mis18α and PLK1 binding were required to activate Mis18α-Mis18ß and promote Mis18 complex-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance.

Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80.

Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.

The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

Glucose fluctuations aggravate cardiomyocyte apoptosis by enhancing the interaction between Txnip and Akt.

BACKGROUND: Glucose fluctuations may be involved in the pathophysiological process of cardiomyocyte apoptosis, but the exact mechanism remains elusive. This study focused on exploring the mechanisms related to glucose fluctuation-induced cardiomyocyte apoptosis. METHODS: Diabetic rats established via an injection of streptozotocin were randomized to five groups: the controlled diabetic (CD) group, the uncontrolled diabetic (UD) group, the glucose fluctuated diabetic (GFD) group, the GFD group rats with the injection of 0.9% sodium chloride (NaCl) (GFD + NaCl) and the GFD group rats with the injection of N-acetyl-L-cysteine (NAC) (GFD + NAC). Twelve weeks later, cardiac function and apoptosis related protein expressions were tested. Proteomic analysis was performed to further analyze the differential protein expression pattern of CD and GFD. RESULTS: The left ventricular ejection fraction levels and fractional shortening levels were decreased in the GFD group, compared with those in the CD and UD groups. Positive cells tested by DAB-TUNEL were increased in the GFD group, compared with those in the CD group. The expression of Bcl-2 was decreased, but the expressions of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in response to glucose fluctuations. Compared with CD, there were 527 upregulated and 152 downregulated proteins in GFD group. Txnip was one of the differentially expressed proteins related to oxidative stress response. The Txnip expression was increased in the GFD group, while the Akt phosphorylation level was decreased. The interaction between Txnip and Akt was enhanced when blood glucose fluctuated. Moreover, the application of NAC partially reversed glucose fluctuations-induced cardiomyocyte apoptosis. CONCLUSIONS: Glucose fluctuations lead to cardiomyocyte apoptosis by up-regulating Txnip expression and enhancing Txnip-Akt interaction.

Identifying and validating the roles of the cuproptosis-related gene DKC1 in cancer with a focus on esophageal carcinoma.

BACKGROUND: Esophageal cancer is a common malignancy of the digestive tract. Despite remarkable advancements in its treatment, the overall prognosis for patients remains poor. Cuproptosis is a form of programmed cell death that affects the malignant progression of tumors. This study aimed to examine the impact of the cuproptosis-associated gene DKC1 on the malignant progression of esophageal cancer. METHODS: Clinical and RNA sequencing data of patients with esophageal cancer were extracted from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis was used to identify the differentially expressed genes related to cuproptosis that are associated with prognosis. We then validated the difference in the expression of DKC1 between tumor and normal tissues via three-dimensional multiomics difference analysis. Subsequently, we investigated the association between DKC1 expression and the tumor microenvironment by employing the TIMER2.0 algorithm, which was further validated in 96 single-cell datasets obtained from the TISCH database. Additionally, the functional role of DKC1 in pancarcinoma was assessed through GSEA. Furthermore, a comprehensive pancancer survival map was constructed, and the expression of DKC1 was verified in various molecular subtypes. By utilizing the CellMiner, GDSC, and CTRP databases, we successfully established a connection between DKC1 and drug sensitivity. Finally, the involvement of DKC1 in the progression of esophageal cancer was investigated through in vivo and in vitro experiments. RESULTS: In this study, we identified a copper death-related gene, DKC1, in esophageal cancer. Furthermore, we observed varying levels of DKC1 expression across different tumor types. Additionally, we conducted an analysis to determine the correlation between DKC1 expression and clinical features, revealing its association with common cell cycle pathways and multiple metabolic pathways. Notably, high DKC1 expression was found to indicate poor prognosis in patients with various tumors and to influence drug sensitivity. Moreover, our investigation revealed significant associations between DKC1 expression and the expression of molecules involved in immune regulation and infiltration of lymphocyte subtypes. Ultimately, the increased expression of DKC1 in esophageal cancer tissues was verified using clinical tissue samples. Furthermore, DKC1-mediated promotion of esophageal cancer cell proliferation and migration was confirmed through both in vitro and in vivo experiments. Additionally, it is plausible that DKC1 may play a role in the regulation of cuproptosis. CONCLUSION: In this study, we conducted a systematic analysis of DKC1 and its regulatory factors and experimentally validated its excellent diagnostic and prognostic abilities in various cancers. Further research indicated that DKC1 may reshape the tumor microenvironment (TME), highlighting the potential of DKC1-based cancer treatment and its usefulness in predicting the response to chemotherapy.

A region-confined PROTAC nanoplatform for spatiotemporally tunable protein degradation and enhanced cancer therapy.

The antitumor performance of PROteolysis-TArgeting Chimeras (PROTACs) is limited by its insufficient tumor specificity and poor pharmacokinetics. These disadvantages are further compounded by tumor heterogeneity, especially the presence of cancer stem-like cells, which drive tumor growth and relapse. Herein, we design a region-confined PROTAC nanoplatform that integrates both reactive oxygen species (ROS)-activatable and hypoxia-responsive PROTAC prodrugs for the precise manipulation of bromodomain and extraterminal protein 4 expression and tumor eradication. These PROTAC nanoparticles selectively accumulate within and penetrate deep into tumors via response to matrix metalloproteinase-2. Photoactivity is then reactivated in response to the acidic intracellular milieu and the PROTAC is discharged due to the ROS generated via photodynamic therapy specifically within the normoxic microenvironment. Moreover, the latent hypoxia-responsive PROTAC prodrug is restored in hypoxic cancer stem-like cells overexpressing nitroreductase. Here, we show the ability of region-confined PROTAC nanoplatform to effectively degrade BRD4 in both normoxic and hypoxic environments, markedly hindering tumor progression in breast and head-neck tumor models.

Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion.

Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.

eIF4E-independent translation is largely eIF3d-dependent.

Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.

High Expression of SRSF10 Promotes Colorectal Cancer Progression by Aberrant Alternative Splicing of RFC5.

BACKGROUND: Colorectal cancer (CRC) remains a global health concern with persistently high incidence and mortality rates. However, the specific pathogenesis of CRC remains poorly understood. This study aims to investigate the role and pathogenesis of serine and arginine rich splicing factor 10 (SRSF10) in colorectal cancer. METHODS: Bioinformatics analysis was employed to predict SRSF10 gene expression in CRC patients. Functional experiments involving SRSF10 knockdown and overexpression were conducted using CCK8, transwell, scratch assay, and flow cytometry. Additionally, the PRIdictor website was utilized to predict the SRSF10 interaction site with RFC5. The identification of different transcripts of SRSF10-acting RFC5 pre-mRNA was achieved through agarose gel electrophoresis. RESULT: The knockdown of SRSF10 inhibited the proliferation and migration ability of CRC cells, while promoting apoptosis and altering the DNA replication of CRC cells. Conversely, when SRSF10 was highly expressed, it enhanced the proliferation and migration ability of CRC cells and caused changes in the cell cycle of colorectal cancer cells. This study revealed a change in the replicating factor C subunit 5 (RFC5) gene in colorectal cancer cells following SRSF10 knockdown. Furthermore, it was confirmed that SRSF10 increased RFC5 exon2-AS1(S) transcription variants, thereby promoting the development of colorectal cancer through AS1 exclusion to exon 2 of RFC5. CONCLUSION: In summary, this study demonstrates that SRSF10 promotes the progression of colorectal cancer by generating an aberrantly spliced exclusion isoform of AS1 within RFC5 exon 2. These findings suggest that SRSF10 could serve as a crucial target for the clinical diagnosis and treatment of CRC.

Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.

Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.

An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

Case report: A mesenchymal chondrosarcoma with alternative HEY1::NCOA2 fusions in the sella turcica.

Introduction: Mesenchymal chondrosarcoma (MCS) is a rare subtype of chondrosarcoma that occurs at widespread anatomical locations, such as bone, soft tissue, and intracranial sites. The central nervous system (CNS) is one of the most common origins of extraosseous MCS. However, alternative HEY1::NCOA2 fusions have not been reported in this tumor. Case report: We report a case of intracranial MCS with HEY1::NCOA2 rearrangement. A 52-year-old woman presented with a 15-mm calcified mass around the sella turcica. She initially underwent transsphenoidal surgery for tumor resection and then additional resections for five local recurrences over 5 years. Histologically, the tumor was composed of small round to spindle-shaped cells admixed with well-differentiated hyaline cartilaginous islands. A hemangiopericytoma-like vascular pattern and small sinusoid-like vessels were also observed. RNA sequencing using RNA extracted from formalin-fixed paraffin-embedded samples from the last operation revealed two alternative variants of the HEY1::NCOA2 fusion: HEY1(ex4)::NCOA2 (ex13) and HEY1(ex4)::NCOA2(ex14). Both variants were confirmed as in-frame fusions using reverse transcription-polymerase chain reaction. Discussion: Cartilaginous components were often not apparent during the recurrences. In addition to the non-typical pathological finding, the correct diagnosis was hampered by the poor RNA quality of the surgical specimens and non-specific STAT6 nuclear staining. Conclusion: This is the first reported case of intracranial MCS with an alternative HEY1::NCOA2 fusion.

Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53.

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.

Targeting of mutant-p53 and MYC as a novel strategy to inhibit oncogenic SPAG5 activity in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient's outcome, and reliable biomarkers for effective anticancer therapies.

Hippo signaling pathway regulates Ebola virus transcription and egress.

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo "ON") where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is "OFF", unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses.

Arl2 GTPase associates with the centrosomal protein Cdk5rap2 to regulate cortical development via microtubule organization.

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.

FIGNL1-FIRRM is essential for meiotic recombination and prevents DNA damage-independent RAD51 and DMC1 loading.

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.

Whole-exome sequencing revealed a novel mutation of the ALMS1 gene in a Chinese family with Alström syndrome: a case report.

BACKGROUND: Alström syndrome (AS) is a rare autosomal recessive disorder that leads to multiple organ fibrosis and failure. Precise diagnosis from the clinical symptoms is challenging due to its highly variabilities and its frequent confusion with other ciliopathies and genetic diseases. Currently, mutations in the ALMS1 gene have been reported as a major cause of AS, thus, it is crucial to focus on the detection and discovery of ALMS1 mutations. CASE PRESENTATION: We present a case of a 13-year-old Chinese boy weighing 70 kg and standing 168 cm tall. He has two younger brothers. Their parents hail from different ancestral homes in eastern and northern China. The patient's primary clinical findings included visual impairment at the age of four and progressive hearing loss starting at the age of ten. Subsequently, at the age of twelve, the patient developed hyperlipidaemia and hyperinsulinemia. Ultrasonographic findings indicated the presence of gallstones and mild fatty liver. His Body Mass Index (BMI) significantly increased to 25 kg/m2 (ref: 18.5-23.9 kg/m2). Additionally, echocardiography revealed mild mitral and tricuspid regurgitation. Ultimately, Whole Exome Sequencing (WES) identified a new missense mutation in the ALMS1 gene (NG_011690.1 (NM_015120): c.9536G > A (p.R3179Q)). This missense mutation generated an aberrant splicer and disrupted the stability and hydrophobicity of proteins, which preliminarily determined as " likely pathogenic". Therefore, considering all the above symptoms and molecular analysis, we deduced that the patient was diagnosed with AS according to the guidelines. We recommended that he continue wearing glasses and undergo an annual physical examination. CONCLUSION: In this case report, we report a novel homozygous ALMS1 mutation associated with AS in the Chinese population, which expands the mutation spectrum of ALMS1. Genetic testing indeed should be incorporated into the diagnosis of syndromic deafness, as it can help avoid misdiagnoses of AS. While there is no specific treatment for AS, early diagnosis and intervention can alleviate the progression of some symptoms and improve patients' quality of life.

The BET PROTAC inhibitor GNE-987 displays anti-tumor effects by targeting super-enhancers regulated gene in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.