Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism.

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer.

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.

Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.

The protein-DNA complexes that compose the end of mammalian chromosomes-telomeres-serve to stabilize linear genomic DNA and are involved in cellular and organismal aging. One mechanism that protects telomeres from premature degradation is the formation of structures called t-loops, in which the single-stranded 3' overhang present at the terminal end of telomeres loops back and invades medial double-stranded telomeric DNA. We identified looped structures formed between terminal chromosome ends and interstitial telomeric sequences (ITSs), which are found throughout the human genome, that we have termed interstitial telomeric loops (ITLs). While they form in a TRF2-dependent manner similar to t-loops, ITLs further require the physical interaction of TRF2 with the nuclear intermediate filament protein lamin A/C. Our findings suggest that interactions between telomeres and the nucleoskeleton broadly impact genomic integrity, including telomere stability, chromosome structure, and chromosome fragility. Here, we review the roles of TRF2 and lamin A/C in telomere biology and consider how their interaction may relate telomere homeostasis to cellular and organismal aging.

Discriminative motif analysis of high-throughput dataset.

MOTIVATION: High-throughput ChIP-seq studies typically identify thousands of peaks for a single transcription factor (TF). It is common for traditional motif discovery tools to predict motifs that are statistically significant against a naïve background distribution but are of questionable biological relevance. RESULTS: We describe a simple yet effective algorithm for discovering differential motifs between two sequence datasets that is effective in eliminating systematic biases and scalable to large datasets. Tested on 207 ENCODE ChIP-seq datasets, our method identifies correct motifs in 78% of the datasets with known motifs, demonstrating improvement in both accuracy and efficiency compared with DREME, another state-of-art discriminative motif discovery tool. More interestingly, on the remaining more challenging datasets, we identify common technical or biological factors that compromise the motif search results and use advanced features of our tool to control for these factors. We also present case studies demonstrating the ability of our method to detect single base pair differences in DNA specificity of two similar TFs. Lastly, we demonstrate discovery of key TF motifs involved in tissue specification by examination of high-throughput DNase accessibility data. AVAILABILITY: The motifRG package is publically available via the bioconductor repository. CONTACT: yzizhen@fhcrc.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide binding of the basic helix-loop-helix myogenic inhibitor musculin has substantial overlap with MyoD: implications for buffering activity.

BACKGROUND: Musculin (MSC) is a basic helix-loop-helix transcription factor that inhibits myogenesis during normal development and contributes to the differentiation defect in rhabdomyosarcoma. As one of many transcription factors that impede myogenesis, its binding on a genome-wide scale relative to the widespread binding of the myogenic factor MyoD is unknown. METHODS: Chromatin immunoprecipitation coupled to high-throughput sequencing was performed for endogenous MSC in rhabdomyosarcoma cells and its binding was compared to that of MyoD in the same type of cells. RESULTS: MSC binds throughout the genome, in a pattern very similar to MyoD. Its binding overlaps strongly with regions enriched for acetylated histone H4, as well as regions that score high for DNase hypersensitivity in human myoblasts. In contrast to MyoD, MSC has a more relaxed binding sequence preference in the nucleotides that flank the core E-box motif. CONCLUSIONS: The myogenic inhibitor MSC binds throughout the genome of rhabdomyosarcoma cells, in a pattern highly similar to that of MyoD, suggesting a broad role in buffering the activity of MyoD in development and rhabdomyosarcomas.

Comparison of genome-wide binding of MyoD in normal human myogenic cells and rhabdomyosarcomas identifies regional and local suppression of promyogenic transcription factors.

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas.

miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells.

BACKGROUND: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state. METHODS: Potential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. RESULTS: Modulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. CONCLUSIONS: These findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation).

Polycomb-mediated repression during terminal differentiation: what don't you want to be when you grow up?

Chromatin-modifying enzymes are known to be critical components for the correct differentiation of embryonic stem cells into specific lineages, such as neurons. Recently, the role of Polycomb group proteins has been studied in the specification and differentiation of muscle stem cells. In this perspective, we review a recent study by Juan and colleagues (pp. 789-794) in Genes & Development of the role of the polycomb group protein Ezh2 in muscle stem cells, and discuss the implications for general lineage restriction.

Genome-wide transcription factor binding: beyond direct target regulation.

The binding of transcription factors to specific DNA target sequences is the fundamental basis of gene regulatory networks. Chromatin immunoprecipitation combined with DNA tiling arrays or high-throughput sequencing (ChIP-chip and ChIP-seq, respectively) has been used in many recent studies that detail the binding sites of various transcription factors. Surprisingly, data from a variety of model organisms and tissues have demonstrated that transcription factors vary greatly in their number of genomic binding sites, and that binding events can significantly exceed the number of known or possible direct gene targets. Thus, current understanding of transcription factor function must expand to encompass what role, if any, binding might have outside of direct transcriptional target regulation. In this review, we discuss the biological significance of genome-wide binding of transcription factors and present models that can account for this phenomenon.

Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming.

Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high-throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro-selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.

Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons.

Little is known about the molecular development and heterogeneity of callosal projection neurons (CPN), cortical commissural neurons that connect homotopic regions of the two cerebral hemispheres via the corpus callosum and that are critical for bilateral integration of cortical information. Here we report on the identification of a series of genes that individually and in combination define CPN and novel CPN subpopulations during embryonic and postnatal development. We used in situ hybridization analysis, immunocytochemistry, and retrograde labeling to define the layer-specific and neuron-type-specific distribution of these newly identified CPN genes across different stages of maturation. We demonstrate that a subset of these genes (e.g., Hspb3 and Lpl) appear specific to all CPN (in layers II/III and V-VI), whereas others (e.g., Nectin-3, Plexin-D1, and Dkk3) discriminate between CPN of the deep layers and those of the upper layers. Furthermore, the data show that several genes finely subdivide CPN within individual layers and appear to label CPN subpopulations that have not been described previously using anatomical or morphological criteria. The genes identified here likely reflect the existence of distinct programs of gene expression governing the development, maturation, and function of the newly identified subpopulations of CPN. Together, these data define the first set of genes that identify and molecularly subcategorize distinct populations of callosal projection neurons, often located in distinct subdivisions of the canonical cortical laminae.

MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state.

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, we have sought to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. In this study, we show that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. Our studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD:E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells.