Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes1-3. Interphase chromosomes are not positioned randomly within the nucleus but instead adopt preferred conformations4-7. Disparate DNA elements co-localize into functionally defined aggregates or “factories” for transcription8 and DNA replication9. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope10-12. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among tRNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

We present single-cell combinatorial indexed Hi-C (sciHi-C), which applies the concept of combinatorial cellular indexing to chromosome conformation capture. In this proof-of-concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karytoypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0728-8) contains supplementary material, which is available to authorized users.

High-throughput methods based on chromosome conformation capture (3C) have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes (REs). Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts that uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution compared to Hi-C. Coupling this method with DNA capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 lincRNA (long intergenic noncoding RNA) promoters in two human cell lines, thereby revealing that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the polycomb repressive complex. Our results provide the first glimpse of a cell type-specific 3D organization of lincRNA genes.

Mutations in Neutrophils and monocytesNeutrophils (also known as "polymorphonuclear leukocytes" and "granulocytes") are terminally differentiated cellular components of the innate immune system that constitute about 35% to 75% of the population of peripheral leukocytes. 1 They kill bacterial and fungal pathogens through phagocytosis and are armed with an arsenal of proteases, antimicrobial peptides, and reactive oxygen species. 2 They also participate in the inflammatory response and produce cytokines, eicosanoids, and other signaling molecules. 3 Monocytes, constituting about 5% to 10% of peripheral leukocytes, are the circulating progenitors of tissue macrophages and dendritic cells and arise in the bone marrow from common myeloid progenitors shared with neutrophils. 4 "Neutropenia" refers to a deficiency in numbers of neutrophils. The normal neutrophil count fluctuates and varies across human populations and within individuals in response to stress and infection but typically well exceeds 1500/L 5 , and neutropenia is usually categorized as severe when the cell count is below 500/L. Common causes of neutropenia include cancer chemotherapy, autoimmune diseases, drug reactions, and hereditary disorders. 6 Among the latter, neutropenia may occur as one component of a number of inherited syndromes diversely featuring morphologic abnormalities of neutrophils, immunodeficiency, involvement of other lineages or pancytopenia, metabolic abnormalities, and systemic findings. This review, however, focuses on the 2 primary genetic forms of neutropenia: cyclic neutropenia (also known as "cyclic hematopoiesis") and the Kostmann syndrome of infantile agranulocytosis, more commonly referred to as "severe congenital neutropenia" (SCN), where mutations of the ELA2 gene, encoding the neutrophil granule serine protease, neutrophil elastase (NE), have proved to be the nearly exclusive or most common cause, respectively. Cyclic neutropeniaIn cyclic neutropenia, the peripheral-blood neutrophil and monocyte counts oscillate in opposite phase to one another with an average 21-day frequency. 6 Peak neutrophil counts tend toward somewhat subnormal values, although they can also be in the normal range. Infections, including aphthous stomatitis, periodontitis, and typhlitis, can arise during the nadir of the cycle, when the neutrophil count drops below 500/L and approaches zero. The infectious flora may differ from what is encountered with acquired neutropenia, suggesting a functional deficiency in neutrophils extending beyond that of low numbers. Cyclic neutropenia is transmitted by autosomal dominant genetics but, as with other often lethal dominant disorders, sporadic cases commonly arise from new germ line mutations. In a remarkable anecdote, a girl with the disease served as a hematopoietic stem cell donor for her sister, who did not have cyclic neutropenia but who was rather suffering from acute lymphoblastic leukemia 7 ; the bone marrow transplantation cured the sibling of leukemia, but it transferred cyclic hematopoiesis to her, ...

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) beta-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

The growth factor independent 1 (Gfi1) transcriptional regulator oncoprotein plays a crucial role in hematopoietic, inner ear, and pulmonary neuroendocrine cell development and governs cell processes as diverse as selfrenewal of hematopoietic stem cells, proliferation, apoptosis, differentiation, cell fate specification, and oncogenesis. However, the molecular basis of its transcriptional functions has remained elusive. Here we show that Gfi1 recruits the histone lysine methyltransferase G9a and the histone deacetylase 1 (HDAC1) in order to modify the chromatin of genes targeted for repression by Gfi1. G9a and HDAC1 are both in a repressive complex assembled by Gfi1. Endogenous Gfi1 colocalizes with G9a, HDAC1, and K9-dimethylated histone H3. Gfi1 associates with G9a and HDAC1 on the promoter of the cell cycle regulator p21 Cip/WAF1, resulting in an increase in K9 dimethylation at histone H3. Silencing of Gfi1 expression in myeloid cells reverses G9a and HDAC1 recruitment to p21Cip/WAF1 and elevates its expression. These findings highlight the role of epigenetics in the regulation of development and oncogenesis by Gfi1.