Faculty Bibliography
Searched for:
in-biosketch:yes
person:skokj01
Total Results:
103
4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments
4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.
PMCID:4777514
PMID: 26938081
ISSN: 1553-7358
CID: 2009402
miRNAs are critical for the regulation of RAG expression and secondary Ig rearrangement in peripheral B lymphocytes [Meeting Abstract]
ISI:000380288302123
ISSN: 1550-6606
CID: 2220232
miRNAs are critical for the regulation of RAG expression and secondary Ig rearrangement in peripheral B lymphocytes [Meeting Abstract]
ISI:000383610400606
ISSN: 1521-4141
CID: 2281742
RAG Off-Target Activity Is in the Loop
The 'off-target' activity of RAG recombinases contributes to mutations and cancer. Recent studies show that the influence of DNA regulatory elements is largely constrained by the formation of chromatin loops and interaction frequencies. Now, the Alt lab has identified major RAG off-target activity bearing similar limitations, with joining events restricted to convergent paired RSS elements in loop domains.
PMCID:4679453
PMID: 26602761
ISSN: 1471-499x
CID: 1856902
Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms
The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.
PMCID:4612095
PMID: 26438359
ISSN: 1540-9538
CID: 1909392
Transcription. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation
Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.
PMCID:4428148
PMID: 25722416
ISSN: 0036-8075
CID: 1474082
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2015:112(5):E458-66.DOI: 10.1073/pnas.1418001112
VH replacement in primary immunoglobulin repertoire diversification
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.
PMCID:4321307
PMID: 25609670
ISSN: 0027-8424
CID: 1440372
Breaking TADs: insights into hierarchical genome organization
PMCID:4624204
PMID: 26111025
ISSN: 1750-192x
CID: 1640992
Long-Range Regulation of V(D)J Recombination
Given their essential role in adaptive immunity, antigen receptor loci have been the focus of analysis for many years and are among a handful of the most well-studied genes in the genome. Their investigation led initially to a detailed knowledge of linear structure and characterization of regulatory elements that confer control of their rearrangement and expression. However, advances in DNA FISH and imaging combined with new molecular approaches that interrogate chromosome conformation have led to a growing appreciation that linear structure is only one aspect of gene regulation and in more recent years, the focus has switched to analyzing the impact of locus conformation and nuclear organization on control of recombination. Despite decades of work and intense effort from numerous labs, we are still left with an incomplete picture of how the assembly of antigen receptor loci is regulated. This chapter summarizes our advances to date and points to areas that need further investigation.
PMCID:4660984
PMID: 26477367
ISSN: 1557-8445
CID: 1809862
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2015:112(5):1256-1258.DOI: 10.1073/pnas.ss11205
PNAS Plus Significance Statements [Editorial]
ISI:000349087700026
ISSN: 0027-8424
CID: 2330672
