Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation.

BACKGROUND: Enhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. RESULTS: Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. CONCLUSIONS: Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

MLL3/MLL4 enzymatic activity shapes DNA replication timing.

Mammalian genomes are replicated in a precise order during S phase, which is cell-type-specific1-3 and correlates with local transcriptional activity2,4-8, chromatin modifications9 and chromatin architecture1,10,11,12. However, the causal relationships between these features and the key regulators of DNA replication timing (RT) are largely unknown. Here, machine learning was applied to quantify chromatin features, including epigenetic marks, histone variants and chromatin architectural factors, best predicting local RT under steady-state and RT changes during early embryonic stem (ES) cell differentiation. About one-third of genome exhibited RT changes during the differentiation. Combined, chromatin features predicted steady-state RT and RT changes with high accuracy. Of these features, histone H3 lysine 4 monomethylation (H3K4me1) catalyzed by MLL3/4 (also known as KMT2C/D) emerged as a top predictor. Loss of Mll3/4 (but not Mll3 alone) or their enzymatic activity resulted in erasure of genome-wide RT dynamics during ES cell differentiation. Sites that normally gain H3K4me1 in a MLL3/4-dependent fashion during the transition failed to transition towards earlier RT, often with transcriptional activation unaffected. Further analysis revealed a requirement for MLL3/4 in promoting DNA replication initiation zones through MCM2 recruitment, providing a direct link for its role in regulating RT. Our results uncover MLL3/4-dependent H3K4me1 as a functional regulator of RT and highlight a causal relationship between the epigenome and RT that is largely uncoupled from transcription. These findings uncover a previously unknown role for MLL3/4-dependent chromatin functions which is likely relevant to the numerous diseases associated with MLL3/4 mutations.

Transcriptional repression upon S phase entry protects genome integrity in pluripotent cells.

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication.

Local injections of ß-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion.

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local ß-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from ß-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local ß-NGF injections. Finally, we demonstrate functional improvements to bone healing following local ß-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate ß-NGF's ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation.

A Drosophila Tumor Suppressor Gene Prevents Tonic TNF Signaling through Receptor N-Glycosylation.

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. Here we report that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. We show that ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. Our results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues.

The transcriptional response to tumorigenic polarity loss in Drosophila.

Loss of polarity correlates with progression of epithelial cancers, but how plasma membrane misorganization drives oncogenic transcriptional events remains unclear. The polarity regulators of the Drosophila Scribble (Scrib) module are potent tumor suppressors and provide a model for mechanistic investigation. RNA profiling of Scrib mutant tumors reveals multiple signatures of neoplasia, including altered metabolism and dedifferentiation. Prominent among these is upregulation of cytokine-like Unpaired (Upd) ligands, which drive tumor overgrowth. We identified a polarity-responsive enhancer in upd3, which is activated in a coincident manner by both JNK-dependent Fos and aPKC-mediated Yki transcription. This enhancer, and Scrib mutant overgrowth in general, are also sensitive to activity of the Polycomb Group (PcG), suggesting that PcG attenuation upon polarity loss potentiates select targets for activation by JNK and Yki. Our results link epithelial organization to signaling and epigenetic regulators that control tissue repair programs, and provide insight into why epithelial polarity is tumor-suppressive.

Endoplasmic Reticulum Stress Activation during Total Knee Arthroplasty.

Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell-stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (-28%, p < .05; -20%, p < .05 respectively) along with an increase in eIF2α phosphorylation (64%, p < .05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, p = .056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, p < .05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, p < .05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated.