Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells

Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.

Biology of hematopoietic stem cells and progenitors: implications for clinical application.

https://www.cell.com/cell/pdf/0092-8674(87)90513-7.pdf

A multiplicity of CCAAT box-binding proteins

In an attempt to find the mechanism by which CpG islands remain free of methylation we have undertaken a detailed examination of the mouse adenine phosphoribosyltransferase (aprt) gene. This housekeeping gene has a CpG island that extends over the gene promoter and includes the first two exons. We show that the island is free of methylation at all CpGs, whereas the flanks are methyated. Detailed patterns of methylation beyond the boundaries of the CpG island vary between cells. In vivo footprinting across the island region shows that three GC boxes clustered at the 5' edge of the CpG island are occupied, most probably by Sp1. No other footprints are detected within the island region. Deletion or mutagenesis of the Sp1 sites causes de novo methylation of the CpG island in a transgenic mouse assay. Thus, the peripherally located Sp1 sites are necessary to keep the aprt island methylation free.

Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island.

The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression.

/pdf/constans-and-the-ccaat-box-binding-complex-share-a-3fyzgjzvwf.pdf

CONSTANS and the CCAAT Box Binding Complex Share a Functionally Important Domain and Interact to Regulate Flowering of Arabidopsis

The sequence 5'TTGGPyCAAT 3' (the 'CCAAT box') is a constituent of the promoter region of many eukaryotic and prokaryotic genes and is believed to play a part in promoter function. A characteristic of the two fetal human globin genes (A gamma and G gamma) is a duplication of a 12-base pair (bp) sequence containing the CCAAT box. Here we report a G----A substitution in the TTG sequence of the distal CCAAT box of the A gamma-globin gene in an individual with the A gamma (Greek) type of hereditary persistence of fetal haemoglobin (HPFH). This represents the first report of a natural mutation of the CCAAT box in a eukaryotic gene. The fact that this transition is associated with inappropriate expression of the A gamma gene in adult life suggests that the CCAAT box (or its surrounding sequences) may have a role in the developmental control of gamma-globin genes.

G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin.

The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSC ex vivo favors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long- and short-term repopulating HSC as detected by an in vivo competitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicate ex vivo.

https://www.pnas.org/content/pnas/96/14/8126.full.pdf

Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin.

Analysis of the megakaryocyte glycoprotein IX promoter identifies positive and negative regulatory domains and functional GATA and Ets sites.

During B cell differentiation, transcription of the J chain gene is initiated. To determine the regulatory mechanism involved, we have analyzed the structure of the J chain gene in cell lines representing successive stages in B cell development. Comparison of restriction sites showed that the J chain gene does not require a rearrangement of DNA for expression; cleavage sites present in embryonic J chain DNA were preserved through all the subsequent differentiative steps. However, comparison of 5-methylcytosine contents showed that J chain gene expression correlates with a loss of methyl groups. The J chain gene was heavily methylated in cell types not expressing J chain (i.e., embryo and lymphomas representative of immature and mature B cells) and significantly less methylated in cell lines representative of antigen-stimulated lymphocytes synthesizing J chain. These changes in J chain gene methylation represent a specific differentiation-induced response. Analyses of the heavy chain C mu and C gamma 2b genes, which are expressed at earlier and later stages than the J chain gene, showed that the C mu sequences were undermethylated in all cell types examined whereas the C gamma 2b sequences were undermethylated only in cell lines expressing the IgG2b protein. The primary encounter of a B cell with antigen must therefore trigger events that effect J chain gene transcription through a mechanism involving loss of methyl groups from cytosine.

https://www.jstor.org/stable/pdfplus/10451.pdf

Expression of the J chain gene during B cell differentiation is inversely correlated with DNA methylation.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

Mayumi Yagi

Papers

G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin.

Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin.

Analysis of the megakaryocyte glycoprotein IX promoter identifies positive and negative regulatory domains and functional GATA and Ets sites.

Expression of the J chain gene during B cell differentiation is inversely correlated with DNA methylation.

Chromatin structure and developmental expression of the human alpha-globin cluster.