Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation.

During development, H3K9me3 heterochromatin is dynamically rearranged, silencing repeat elements and protein coding genes to restrict cell identity. Enhancer of Rudimentary Homolog (ERH) is an evolutionarily conserved protein originally characterized in fission yeast and recently shown to be required for H3K9me3 maintenance in human fibroblasts, but its function during development remains unknown. Here, we show that ERH is required for proper segregation of the inner cell mass and trophectoderm cell lineages during mouse development by repressing totipotent and alternative lineage programs.

Diverse heterochromatin states restricting cell identity and reprogramming.

Heterochromatin is defined as a chromosomal domain harboring repressive H3K9me2/3 or H3K27me3 histone modifications and relevant factors that physically compact the chromatin. Heterochromatin can restrict where transcription factors bind, providing a barrier to gene activation and changes in cell identity. While heterochromatin thus helps maintain cell differentiation, it presents a barrier to overcome during efforts to reprogram cells for biomedical purposes.

A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling.

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPβ. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3.

Two-parameter single-molecule analysis for measurement of chromatin mobility.

This protocol provides a two-parameter analysis of single-molecule tracking (SMT) trajectories of Halo-tagged histones in living adherent cell lines and unveils a chromatin mobility landscape composed of five chromatin types, ranging from low to high mobility. When the analysis is applied to Halo-tagged, chromatin-binding proteins, it associates chromatin interaction properties with known functions in a way that previously used SMT parameters did not. For complete information on the use and execution of this protocol, please refer to Lerner et al.

Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin.

Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes.

Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin.

Rapid monoisotopic cisplatin based barcoding for multiplexed mass cytometry.

Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously.

Sample Preparation for Mass Cytometry Analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis.

MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers.

The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation.

Wood is mainly composed of secondary walls, which constitute the most abundant stored carbon produced by vascular plants. Understanding the molecular mechanisms controlling secondary wall deposition during wood formation is not only an important issue in plant biology but also critical for providing molecular tools to custom-design wood composition suited for diverse end uses. Past molecular and genetic studies have revealed a transcriptional network encompassing a group of wood-associated NAC and MYB transcription factors that are involved in the regulation of the secondary wall biosynthetic program during wood formation in poplar trees.

Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar.

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls.

Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors.

The bulk of grass biomass potentially useful for cellulose-based biofuel production is the remains of secondary wall-containing sclerenchymatous fibers. Hence, it is important to uncover the molecular mechanisms underlying the regulation of secondary wall thickening in grass species. So far, little is known about the transcriptional regulatory switches responsible for the activation of the secondary wall biosynthetic program in grass species.

Secondary wall NAC binding element (SNBE), a key cis-acting element required for target gene activation by secondary wall NAC master switches.

The biosynthesis of secondary walls in vascular plants requires the coordinated regulation of a suite of biosynthetic genes, and this coordination has recently been shown to be executed by the secondary wall NAC (SWN)-mediated transcriptional network. In Arabidopsis, five SWNs, including SND1, NST1/2 and VND6/7, function as master transcriptional switches to activate their common targets and consequently the secondary wall biosynthetic program. A recent report by Zhong et al.

The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis.

Dicot wood is mainly composed of cellulose, xylan and lignin, and its formation requires the coordinated regulation of their biosynthesis. In this report, we demonstrate that the poplar wood-associated MYB transcriptional activators, PtrMYB3 and PtrMYB20, activate the biosynthetic pathways of cellulose, xylan and lignin when overexpressed in Arabidopsis and they are also able to activate the promoter activities of poplar wood biosynthetic genes. We also show that PtrMYB3 and PtrMYB20 are functional orthologs of Arabidopsis MYB46 and MYB83, and their expression is directly activated by poplar PtrWND2, suggesting their involvement in the regulation of wood formation in poplar.

MYB83 is a direct target of SND1 and acts redundantly with MYB46 in the regulation of secondary cell wall biosynthesis in Arabidopsis.

It has been proposed that the transcriptional regulation of secondary wall biosynthesis in Arabidopsis is controlled by a transcriptional network mediated by SND1 and its close homologs. Uncovering all the transcription factors and deciphering their interrelationships in the network are essential for our understanding of the molecular mechanisms underlying the transcriptional regulation of biosynthesis of secondary walls, the major constituent of wood and fibers. Here, we present functional evidence that the MYB83 transcription factor is another molecular switch in the SND1-mediated transcriptional network regulating secondary wall biosynthesis.

A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis.

SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening.