BACKGROUND: Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive.
METHODS: In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining.
RESULTS: USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells.
CONCLUSIONS: These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.

The nucleosome remodeling and deacetylase (NuRD) complex plays a pivotal role in chromatin regulation and transcriptional repression. In mice, methyl-CpG binding domain 3 isoform C (MBD3C) interacts specifically with the histone H3 binding protein WD repeat-containing protein 5 (WDR5) and forms the WDR5-MBD3C/Norde complex. Despite the functional significance of this interaction on embryonic stem cell gene regulation, the molecular mechanism underlying MBD3C recognition by WDR5 remains elusive. Here, we determined the crystal structure of WDR5 in complex with the peptide (residues 40-51) derived from the MBD3C protein at a resolution of 1.9 Å. Structural analysis revealed that MBD3C utilizes a unique binding mode to interact with WDR5, wherein MBD3C Arg43 and Phe47 are involved in recognizing the WDR5-interacting (WIN) site and Tyr191-related B site on the small surface of WDR5, respectively. Notably, the binding induces a ∼91° rotation of WDR5 Tyr191, generating the hydrophobic B site. Furthermore, mutation experiments combined with isothermal titration calorimetry (ITC) assays confirmed the importance of both Arg43 and Phe47 in mediating WDR5 binding affinity. By determining structures of various peptides bound to WDR5, we demonstrated that the WDR5 WIN site and B site can be concurrently recognized by WIN motif peptides containing \'\'Arg-Cies/Ser-Arg-Val-Phe\'\' consensus sequence. Overall, this study reveals the structural basis for the formation of the WDR5-MBD3C subcomplex and provides new insights into the recognition mode of WDR5 for the WIN motif. Moreover, these findings shed light on structural-based designs of WDR5-targeted anti-cancer small molecule inhibitors or peptide-mimic drugs.

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear protein that attaches negatively charged poly (ADP-ribose) (PAR) to itself and other target proteins. While its function in DNA damage repair is well established, its role in target chromatin recognition and regulation of gene expression remains to be better understood. This study showed that PARP1 interacts with SET1/MLL complexes by binding directly to WDR5. Notably, although PARP1 does not modulate WDR5 PARylation or the global level of H3K4 methylation, it exerts locus-specific effects on WDR5 binding and H3K4 methylation. Interestingly, PARP1 and WDR5 show extensive co-localization on chromatin, with WDR5 facilitating the recognition and expression of target genes regulated by PARP1. Furthermore, we demonstrated that inhibition of the WDR5 Win site impedes the interaction between PARP1 and WDR5, thereby inhibiting PARP1 from binding to target genes. Finally, the combined inhibition of the WDR5 Win site and PARP shows a profound inhibitory effect on the proliferation of cancer cells. These findings illuminate intricate mechanisms underlying chromatin recognition, gene transcription, and tumorigenesis, shedding light on previously unrecognized roles of PARP1 and WDR5 in these processes.

Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear.
Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter.
AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression.
We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.

The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.

WDR5 is a highly conserved protein that performs multiple scaffolding functions in the context of chromatin. However, efforts to understand the function of WDR5 in normal tissues physiologically are quite limited so far. In our study, we explored the function of Wdr5 in erythropoiesis and hematopoiesis by using a hematopoietic-specific Wdr5 knockout mouse model. We found that loss of Wdr5 mediated by Vav-iCre leads to embryonic lethality with defective erythropoiesis. In addition, Wdr5-deficiency completely impairs the hematopoietic stem and progenitor cells function and might alter the immunophenotype of these stem cells and progenitors by decreasing c-Kit expression. Collectively, we identified the pivotal role of Wdr5 in fetal hematopoiesis and erythropoiesis as the de novo findings.

Myeloid-derived suppressor cells (MDSCs) are a population of immature bone marrow cells that accumulate in large numbers in the spleen, peripheral blood, bone marrow, lymph nodes, and local and metastatic foci of tumors. C/EBP homologous protein (CHOP) and CCAAT/enhancer binding protein β (C/EBPβ) play key roles in regulating the immunosuppressive function and differentiation of MDSCs. Our study revealed that the long noncoding RNA Lnc-17Rik was able to promote immunosuppression in tumors by facilitating the activation and expression of key genes involved in MDSC differentiation. Lnc-17Rik was shown to directly interact with CHOP and C/EBPβ LIP to facilitate their dissociation from the transcriptional repressor complex involving C/EBP LAP/LIP/CHOP. Moreover, Lnc-17Rik increased the association of WD repeat-containing protein 5 (WDR5) with C/EBP LAP, promoting H3K4me3 enrichment in the promoter regions of arginase 1 (Arg-1), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2) and NADPH oxidase 2 (NOX2) to enhance the expression of these genes. Furthermore, using a CD45 chimeric model we confirmed that Lnc-17Rik promoted the differentiation of monocytic (M)-MDSCs in vivo with the introduction of Lnc-17Rik-overexpressing MDSCs shown to promote tumor growth as a result of enhancing their immunosuppressive function. Notably, human Lnc-17Rik is highly homologous to mouse Lnc-17Rik and fulfills similar functions in human MDSC-like cells. In addition, we also found a high level of Lnc-17Rik in peripheral blood MDSC of patients with esophageal cancer. These findings suggest that Lnc-17Rik plays an important role in controlling the immunosuppressive function of MDSCs in the tumor environment and may further serve as a potential therapeutic target for patients with esophageal cancer.

BACKGROUND: The aberrantly increased proliferation and migration of vascular smooth muscle cells (VSMCs) was critically associated with atherosclerosis (AS) progression. MiR-197-3p has been confirmed to regulate various biological processes, such as tumorigenesis; however, whether miR-197-3p is involved with the pathological development of AS remains largely unknown.
METHODS: The serum levels of miR-197-3p in AS patients and healthy donors were determined by polymerase chain reaction (PCR) assay. The transfection efficacies of miR-197-3p mimic or inhibitor in VSMCs were evaluated by PCR assay. The effects of miR-197-3p on VSMC proliferation and migration were determined by EdU cell proliferation and Traswell migration assays. Western blotting was conducted to evaluate the effect of miR-197-3p on WDR5 expression in VSMCs.
RESULTS: In the present study, we found that the expression of miR-197-3p was decreased in the serum of AS patients compared to healthy donors. Overexpression of miR-197-3p inhibited the proliferation and migration of VSMCs, while silencing miR-197-3p showed opposite effects. Mechanistical study revealed that WD Repeat Domain 5 (WDR5) was a target of miR-197-3p. Moreover, miR-197-3p was downregulated in VSMCs upon IL6 treatment and inhibited IL6-induced proliferation and migration in VSMCs.
CONCLUSIONS: These findings indicate that miR-197-3p could serve as a promising diagnostic marker for AS and that targeting IL6/miR-197-3p/WDR5 axis might be a potential approach to treat AS.

T-cell acute lymphoblastic leukemia (T-ALL) is a common hematologic malignancy. Based on the data from GSE66638 and GSE141140, T-ALL patients depicted a higher USP44 level. However, its role in T-ALL is still unclear. In the present study, we investigated the role of USP44 in T-ALL growth. USP44 overexpression elevated the proliferation of CCRF-CEM cells, while USP44 knockdown suppressed the proliferation of Jurkat and MOLT-4 cells. In addition, USP44 accelerated the cell cycle progression, with boosted cyclinD and PCNA levels. However, USP44 knockdown induced apoptosis in Jurkat and MOLT-4 cells, with an upheaval among cleaved caspase-3 and PARP levels. Mechanistically, USP44 co-localized and interacted with WDR5, leading to the repression of its ubiquitination and degradation. Interestingly, WDR5 overexpression abolished the apoptosis induced by USP44 knockdown. Consistently, the in vivo study revealed that USP44 knockdown restricted the leukemic engraftments in the bone marrow and spleens and reduced the infiltration of T-ALL cells in the livers and lungs. In conclusion, this study indicated that USP44 enhanced the growth of T-ALL through interacting with WDR5 and repressing its ubiquitination. This study highlights the potential use of USP44 as a therapeutic target of T-ALL.

Intermittent hypoxia (IH) is a factor involved in the incidence and progression of lung adenocarcinoma (LUAD). Bone marrow-derived bone mesenchymal stem cells (BMSCs)-derived exosomes are related to the promotion of tumor development. The objective of this experiment was to clarify the mechanism of exosomes from BMSCs in promoting the progression of LUAD induced by IH.
This study examined if IH BMSCS-derived exosomes affect the malignancy of LUAD cells in vitro. Dual-luciferase assays were conducted to confirm the target of miR-31-5p with WD repeat domain 5 (WDR5). We further investigated whether or not  exosomal miR-31-5p or WDR5 could regulate epithelial-mesenchymal transition (EMT). We determined the effect of IH exosomes using a tumorigenesis model in vivo.
miR-31-5p entered into LUAD cells via exosomes. MiR-31-5p was greatly upregulated in IH BMSCs-derived exosomes compared with RA exosomes. Increased expression of exosomal miR-31-5p induced by IH was discovered to target WDR5 directly, increased activation of WDR5, and significantly facilitated EMT, thereby promoting LUAD progression.
The promoting effect of IH on LUAD is achieved partly through BMSCs-derived exosomal miR-31-5p triggering WDR5 and promoting EMT.