M. A. Bender

An expansive human regulatory lexicon encoded in transcription factor footprints

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis–regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein–DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

A Complex Chromatin Landscape Revealed by Patterns of Nuclease Sensitivity and Histone Modification within the Mouse β-Globin Locus

ABSTRACT In order to create an extended map of chromatin features within a mammalian multigene locus, we have determined the extent of nuclease sensitivity and the pattern of histone modifications associated with the mouse β-globin genes in adult erythroid tissue. We show that the nuclease-sensitive domain encompasses the β-globin genes along with several flanking olfactory receptor genes that are inactive in erythroid cells. We describe enhancer-blocking or boundary elements on either side of the locus that are bound in vivo by the transcription factor CTCF, but we found that they do not coincide with transitions in nuclease sensitivity flanking the locus or with patterns of histone modifications within it. In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse β-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active β-globin locus and illustrate the complexity of broad structural changes that accompany gene activation.

Reduced β‐Globin Gene Expression in Adult Mice Containing Deletions of Locus Control Region 5′ HS‐2 or 5′ HS‐3

Abstract: To gain insights into the functions of individual DNAse hypersensitive sites within the β globin locus control region (LCR), we deleted the endogenous 5′ HS‐2 and HS‐3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5′ HS‐2 or 5′ HS‐3 reduced the expression of the embryonic ɛy and βh1 globin genes minimally in yolk sac‐derived erythrocytes, but that both knockouts reduced the output of the adult β (β‐Major +β‐Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK‐Neo cassette was retained within either the HS‐2 or HS‐3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK‐Neo was shown to be expressed in an erythroid‐specific fashion when it was retained in the HS‐3 position. These results show that neither 5′ HS‐2 nor HS‐3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult β‐globin gene expression in adult red blood cells. Each site therefore contains some non‐redundant information that contributes to adult globin gene function.

Long-Term Expression of the Human ß-Globin Gene After Retroviral Transfer into Pluripotent Hematopoietic Stem Cells of the Mouse

We have studied the regulation of the human beta-globin gene after retroviral transfer into a variety of transformed and normal hematopoietic cells. After transfer into murine erythroleukemia cells (MEL) expression from the human beta-globin gene responds to inducers of erythroid maturation in parallel to the endogenous murine globin genes. After infection of human BFU-E, RNA expression from the virally-transferred beta-globin gene was measured at 2.5%-5% of the endogenous beta-globin level. The most improved globin vectors can transfer the human beta-globin gene into pluripotent hematopoietic stem cells in mouse bone marrow. Mice reconstituted with infected marrow show human beta-globin RNA and protein expression in peripheral blood cells for over 4 months. In these animals, both myeloid and lymphoid cells carry the integrated provirus at a level of about 1 copy per cell. In serial transplantation experiments, bone marrow from these animals is capable of repopulating secondary and tertiary recipient animals which go on to show long-term human beta-globin expression. Retroviral vectors thus provide a practical way to refine models of globin gene regulation through in vivo tests and to evaluate the feasibility of protocols for gene addition therapy.

Liver and heart MR relaxometry in iron loading: Reproducibility of three methods

To compare the derived T2* values and reproducibility of three methods used to assess iron‐loading in heart and liver.