Tumor microenvironmental determinants of high-risk DCIS progression.

Ductal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8- T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.

Evidence-Based Storytelling for a Strategic Roadmap to Promote Cancer Prevention via Adolescent HPV Vaccination in Northern New England.

Vaccination rates for the human papillomavirus (HPV) among rural youth in northern New England lag those of more urbanized areas. Reasons include a lack of available medical offices, time constraints, perceptions of vaccines and HPV, and, to a smaller degree, delays caused by the COVID-19 pandemic. We have a responsibility to increase vaccinations in these communities. To do so, vaccination experts recommend addressing the three C's of vaccination hesitation: confidence, complacency, and convenience. With this framework as our foundation, in this article we detail a plan to address these important elements, and we add several more C's: clinics, communication, collaboration, community, capacity, and commitment to the list as we discuss the essential pieces-human, infrastructural, and perceptual-needed to create and promote successful, community-supported, school-based HPV vaccination clinics to serve youths aged nine to 18. We then integrate research and storytelling science into an innovative Persuasion Playbook, a guide for local opinion leaders to use in creating evidence-based, pro-vaccine messages on the community level to promote the clinics via evidence-based, pro-vaccination messages.

Epigenetic regulators controlling osteogenic lineage commitment and bone formation.

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.

WWOX promotes osteosarcoma development via upregulation of Myc.

Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.

Disrespectful and inadequate palliative care to transgender persons.

OBJECTIVES: The purpose of this study was to describe disrespectful, inadequate, and abusive care to seriously ill patients who identify as transgender and their partners. METHODS: A cross-sectional mixed methods study was conducted. The sample included 865 nurses, physicians, social workers, and chaplains. Respondents were asked whether they had observed disrespectful, inadequate, or abusive care due to the patient being transgender and to describe such care. RESULTS: Of the 21.3% of participants who reported observing discriminatory care to a transgender patient, 85.3% had observed disrespectful care, 35.9% inadequate care, and 10.3% abusive care. Disrespectful care included insensitivity; rudeness, ridicule, and gossip by staff; not acknowledging or accepting the patient's gender identity or expression; privacy violations; misgendering; and using the incorrect name. Inadequate care included denying, delaying, or rushing care; ignorance of appropriate medical and other care; and marginalizing or ignoring the spouse/partner. SIGNIFICANCE OF RESULTS: These findings illustrate discrimination faced by seriously ill transgender patients and their spouse/partners. Providers who are disrespectful may also deliver inadequate care to transgender patients, which may result in mistrust of providers and the health-care system. Inadequate care due to a patient's or spouse's/partner's gender identity is particularly serious. Dismissing spouses/partners as decision-makers or conferring with biological family members against the patient's wishes may result in unwanted care and constitute a Health Insurance Portability and Accountability Act of 1996 (HIPAA) violation. Institutional policies and practices should be assessed to determine the degree to which they are affirming to both patients and staff, and revised if needed. Federal and state civil rights legislation protecting the LGBTQ+ community are needed, particularly given the rampant transphobic legislation and the majority of states lacking civil rights laws protecting LGBTQ+ people. Training healthcare professionals and staff to become competent and comfortable treating transgender patients is critical to providing optimal care for these seriously ill patients and their spouse/partner.

Multispectral Imaging of Metabolic Fluorophores: Comparing In Vivo and Fresh Ex Vivo Tissue.

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation.

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.

Raising the HPV Vaccination Rate in Rural Northern New England Using Local Opinion Leaders.

The human papillomavirus is associated with a range of cancers. A vaccine introduced in 2006 has dramatically decreased the incidence of these cancers, but Americans still experience over 47,000 new cases of HPV-related cancers each year. The situation is worse in rural areas, where vaccination rates lag the national average, making HPV a significant health disparity issue. This article lays out an evidence-based HPV vaccine-promotion strategy that will serve as part of a campaign to improve health equity in rural northern New England in a process that is repeatable and sustainable. The campaign includes the following elements: partnerships with state departments of health and trusted community opinion leaders, evidence-based storytelling, local social media, traditional media, and school-based pop-up vaccination clinics. Borrowing from marketing and social marketing frameworks and guided by public health perspectives, we begin with psychographic and geodemographic information about our target audience, followed by a discussion about relevant models, frameworks, and research related to persuasive storytelling. We conclude with the outline of a guidebook to foster the creation of persuasive stories as part of a sustainable, replicable HPV vaccination campaign.

Disrespectful and inadequate palliative care to lesbian, gay, and bisexual patients.

OBJECTIVES: The study aims to describe inadequate, disrespectful, and abusive palliative and hospice care received by lesbian, gay, and bisexual (LGB) patients and their spouses/partners due to their sexual orientation or gender identity. METHODS: A national sample of 865 healthcare professionals recruited from palliative and hospice care professional organizations completed an online survey. Respondents were asked to describe their observations of inadequate, disrespectful, or abusive care to LGB patients and their spouses/partners. RESULTS: There were 15.6% who reported observing disrespectful care to LGB patients, 7.3% observed inadequate care, and 1.6% observed abusive care; 43% reported discriminatory care toward the spouses/partners. Disrespectful care to LGB patients included insensitive and judgmental attitudes and behaviors, gossip and ridicule, and disrespect of the spouse/partner. Inadequate care included denial of care; care that was delayed incomplete, or rushed; dismissive or antagonistic treatment; privacy and confidentiality violations; and dismissive treatment of the spouse/partner. SIGNIFICANCE OF RESULTS: These findings provide evidence of discrimination faced by LGB patients and partners while receiving care for serious illness. Hospice and palliative care programs should promote respectful, inclusive, and affirming care for the lesbian, gay, bisexual, transgender, and queer (LGBTQ) community, including policies and practices that are welcoming and supportive to both employees and patients. Staff at all levels should be trained to create safe and respectful environments for LGBTQ patients and their families.

Cbfß Is a Novel Modulator against Osteoarthritis by Maintaining Articular Cartilage Homeostasis through TGF-ß Signaling.

TGF-ß signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-ß signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor ß (Cbfß) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfß in maintaining articular cartilage integrity remains obscure. This study investigated Cbfß as a novel anabolic modulator of TGF-ß signaling and determined its role in articular cartilage homeostasis. Cbfß significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfß rescued Type II collagen (Col2α1) and Runx1 expression in Cbfß-deficient chondrocytes. TGF-ß1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-ß1 treatment in Cbfß-deficient chondrocytes. Cbfß protected Runx1 from proteasomal degradation through Cbfß/Runx1 complex formation. These results indicate that Cbfß is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfß could protect OA development by maintaining the integrity of the TGF-ß signaling pathway in articular cartilage.

Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression.

Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located âˆ¼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

peaksat: an R package for ChIP-seq peak saturation analysis.

BACKGROUND: Epigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types. RESULTS: We developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq. CONCLUSION: peaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.

The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization.

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation.