Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an ∼250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes

Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.

BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations

The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator–containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5′ mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3′ region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions

Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function

Live-Cell Chromosome Dynamics and Outcome of X Chromosome Pairing Events during ES Cell Differentiation

SummaryRandom X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation