Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

A modular open platform for systematic functional studies under physiological conditions.

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.

Prenatal Diagnosis of Fryns Syndrome through Identification of Two Novel Splice Variants in the PIGN Gene-A Case Series.

Fryns syndrome (FS) is a multiple congenital anomaly syndrome with different multisystemic malformations. These include congenital diaphragmatic hernia, pulmonary hypoplasia, and craniofacial dysmorphic features in combination with malformations of the central nervous system such as agenesis of the corpus callosum, cerebellar hypoplasia, and enlarged ventricles. We present a non-consanguineous northern European family with two recurrent cases of FS: a boy with multiple congenital malformations who died at the age of 2.5 months and a female fetus with a complex developmental disorder with similar features in a following pregnancy. Quad whole exome analysis revealed two likely splicing-affecting disease-causing mutations in the PIGN gene: a synonymous mutation c.2619G>A, p.(Leu873=) in the last nucleotide of exon 29 and a 30 bp-deletion c.996_1023+2del (NM_176787.5) protruding into intron 12, with both mutations in trans configuration in the affected patients. Exon skipping resulting from these two variants was confirmed via RNA sequencing. Our molecular and clinical findings identified compound heterozygosity for two novel splice-affecting variants as the underlying pathomechanism for the development of FS in two patients.

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Spatial organization of transcribed eukaryotic genes.

Despite the well-established role of nuclear organization in the regulation of gene expression, little is known about the reverse: how transcription shapes the spatial organization of the genome. Owing to the small sizes of most previously studied genes and the limited resolution of microscopy, the structure and spatial arrangement of a single transcribed gene are still poorly understood. Here we study several long highly expressed genes and demonstrate that they form open-ended transcription loops with polymerases moving along the loops and carrying nascent RNAs. Transcription loops can span across micrometres, resembling lampbrush loops and polytene puffs. The extension and shape of transcription loops suggest their intrinsic stiffness, which we attribute to decoration with multiple voluminous nascent ribonucleoproteins. Our data contradict the model of transcription factories and suggest that although microscopically resolvable transcription loops are specific for long highly expressed genes, the mechanisms underlying their formation could represent a general aspect of eukaryotic transcription.

HP1ß carries an acidic linker domain and requires H3K9me3 for phase separation.

Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1ß. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1ß which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1ß mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1ß with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.