Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Allelic differences between Europeans and Chinese for CREB1 SNPs and their implications in gene expression regulation, hippocampal structure and function, and bipolar disorder susceptibility.

Bipolar disorder (BD) is a polygenic disorder that shares substantial genetic risk factors with major depressive disorder (MDD). Genetic analyses have reported numerous BD susceptibility genes, while some variants, such as single-nucleotide polymorphisms (SNPs) in CACNA1C have been successfully replicated, many others have not and subsequently their effects on the intermediate phenotypes cannot be verified. Here, we studied the MDD-related gene CREB1 in a set of independent BD sample groups of European ancestry (a total of 64,888 subjects) and identified multiple SNPs significantly associated with BD (the most significant being SNP rs6785[A], P=6.32 × 10(-5), odds ratio (OR)=1.090). Risk SNPs were then subjected to further analyses in healthy Europeans for intermediate phenotypes of BD, including hippocampal volume, hippocampal function and cognitive performance. Our results showed that the risk SNPs were significantly associated with hippocampal volume and hippocampal function, with the risk alleles showing a decreased hippocampal volume and diminished activation of the left hippocampus, adding further evidence for their involvement in BD susceptibility. We also found the risk SNPs were strongly associated with CREB1 expression in lymphoblastoid cells (P<0.005) and the prefrontal cortex (P<1.0 × 10(-6)). Remarkably, population genetic analysis indicated that CREB1 displayed striking differences in allele frequencies between continental populations, and the risk alleles were completely absent in East Asian populations. We demonstrated that the regional prevalence of the CREB1 risk alleles in Europeans is likely caused by genetic hitchhiking due to natural selection acting on a nearby gene. Our results suggest that differential population histories due to natural selection on regional populations may lead to genetic heterogeneity of susceptibility to complex diseases, such as BD, and explain inconsistencies in detecting the genetic markers of these diseases among different ethnic populations.

Discovery and replication of dopamine-related gene effects on caudate volume in young and elderly populations (N=1198) using genome-wide search.

The caudate is a subcortical brain structure implicated in many common neurological and psychiatric disorders. To identify specific genes associated with variations in caudate volume, structural magnetic resonance imaging and genome-wide genotypes were acquired from two large cohorts, the Alzheimer's Disease NeuroImaging Initiative (ADNI; N=734) and the Brisbane Adolescent/Young Adult Longitudinal Twin Study (BLTS; N=464). In a preliminary analysis of heritability, around 90% of the variation in caudate volume was due to genetic factors. We then conducted genome-wide association to find common variants that contribute to this relatively high heritability. Replicated genetic association was found for the right caudate volume at single-nucleotide polymorphism rs163030 in the ADNI discovery sample (P=2.36 × 10⁻6) and in the BLTS replication sample (P=0.012). This genetic variation accounted for 2.79 and 1.61% of the trait variance, respectively. The peak of association was found in and around two genes, WDR41 and PDE8B, involved in dopamine signaling and development. In addition, a previously identified mutation in PDE8B causes a rare autosomal-dominant type of striatal degeneration. Searching across both samples offers a rigorous way to screen for genes consistently influencing brain structure at different stages of life. Variants identified here may be relevant to common disorders affecting the caudate.

Regulation of histone gene expression.

Histone genes are expressed during the S phase of the cell cycle. Control is at multiple levels and is mediated by the integration of regulatory signals in response to cell-cycle progression and the onset of differentiation. Much work has been carried out on the H4 gene promoter, which appears to be organized into a series of distinct regulatory elements. The three-dimensional organization of the promoter and, in particular, its spatial relationship with the nuclear matrix scaffold, may be important factors of transcription regulation.

Subtidal gastropods consume sulfur-oxidizing bacteria: evidence from coastal hydrothermal vents.

The black abalone (Haliotis cracherodii), a commercially important shallow-water gastropod common off White Point, Southern California, is found frequently at subtidal hydrothermal vents within mats of filamentous sulfur-oxidizing bacteria. Foraging vent abalones actively consume the bacteria and confine their nightly feeding forays to bacterial mats surrounding the vents. The growth of abalones consuming the sulfur bacteria exceeds that of control individuals consuming microalgae and is comparable to reported growth rates of abalones consuming macroalgae. Thus, off White Point, the black abalone may derive a portion of its nutrition from the subsidy of geothermal energy.

Histone proteins in HeLa S3 cells are synthesized in a cell cycle stage specific manner.

The synthesis of histone proteins in G1 and S phase HeLa S3 cells was examined by two-dimensional electrophoretic fractionation of nuclear and total cellular proteins. Newly synthesized histones were detected only in S phase cells. Histone messenger RNA sequences, as detected by hybridization with cloned human histone genes, were present in the cytoplasm of S phase but not G1 cells.

Activation of histone gene transcription by nonhistone chromosomal phosphoproteins.

Hybridization analysis of RNA transcripts from HeLa S3 cell chromatin to histone complementary DNA indicates that a chromosomal phosphoprotein fraction activates transcription of histone messenger RNA sequences in vitro with chromatin from a phase in the cell cycle when histone genes are normally silent.

Hybridization analysis of histone messenger RNA: association with polyribosomes during the cell cycle.

Hybridization of cell cycle stage-specific polyribosomal RNA's to histone complementary DNA indicates that histone messenger RNA sequences are present on polyribosomes of Hela S3 cells only during the period of DNA replication.

Changes in the stability of a human H3 histone mRNA during the HeLa cell cycle.

A major component of the regulation of histone protein synthesis during the cell cycle is the modulation of the half-life of histone mRNA. We have uncoupled transcriptional and posttranscriptional regulation by using a Drosophila hsp70-human H3 histone fusion gene that produces a marked human H3 histone mRNA upon heat induction. Transcription of this gene can be switched on and off by raising and lowering cell culture temperatures, respectively. HeLa cell lines containing stably integrated copies of the fusion gene were synchronized by double thymidine block. Distinct populations of H3 histone mRNA were produced by heat induction in early S-phase, late S-phase, or G2-phase cells, and the stability of the induced H3 histone mRNA was measured. The H3 histone mRNA induced during early S phase decayed with a half-life of 110 min, whereas the same transcript induced during late S phase had a half-life of 10 to 15 min. The H3 histone mRNA induced in non-S-phase cells is more stable than that induced in late S phase, with a half-life of 40 min. Thus, the stability of histone mRNA is actively regulated throughout the cell cycle. Our results are consistent with an autoregulatory model in which the stability of histone mRNA is determined by the level of free histone protein in the cytoplasm.

Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

Effect of adenovirus infection on expression of human histone genes.

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.

Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization.

Three Cbfa motifs are strategically positioned in the bone-specific rat osteocalcin (rOC) promoter. Sites A and B flank the vitamin D response element in the distal promoter and sites B and C flank a positioned nucleosome in the proximal promoter. The functional significance of each Cbfa element was addressed by mutating individual or multiple Cbfa sites within the context of the -1.1-kb rOC promoter fused to a chloramphenicol acetyltransferase reporter gene. Promoter activity was assayed following transient transfection and after stable genomic integration in ROS 17/2.8 osteoblastic cell lines. We show that all three Cbfa sites are required for maximal basal expression of the rOC promoter. However, the distal sites A and B each contribute significantly more (P < 0.001) to promoter activity than site C. In a genomic context, sites A and B can largely compensate for a mutation at the proximal site C, and paired mutations involving site A (mAB or mAC) result in a far greater loss of activity than the mBC mutation. Strikingly, mutation of the three Cbfa sites leads to abrogation of responsiveness to vitamin D. Vitamin D-enhanced activity is also not observed when sites A and B are mutated. Significantly, related to these losses in transcriptional activity, mutation of the three Cbfa sites results in altered chromatin structure as reflected by loss of DNase I-hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These findings strongly support a multifunctional role for Cbfa factors in regulating gene expression, not only as simple transcriptional transactivators but also by facilitating modifications in promoter architecture and chromatin organization.

runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins.

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.

Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact.

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3' end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3' end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.

Influence of chlorambucil, a bifunctional alkylating agent, on DNA replication and histone gene expression in HeLa S3 cells.

We have examined the influence of chlorambucil, a bifunctional alkylating agent that inhibits cell proliferation, on DNA replication and histone gene expression in exponentially growing HeLa S3 cells. During the period of treatment with chlorambucil (up to 3 days), neither transcription nor translation in general appeared to be affected, but the incorporation of [14C]thymidine into DNA was reduced to 15% of control values by the third day. The appearance of newly synthesized histones and non-histone proteins on chromatin was inhibited with a time course similar to that for inhibition of DNA synthesis. However, the representation of histone messenger RNA sequences in various cellular compartments did not appear to be affected by chlorambucil treatment, in contrast to the loss of histone messenger RNA sequences from polyribosomes following the more rapid inhibition of DNA and histone synthesis by 1-beta-D-arabinofuranosylcytosine or hydroxyurea. The possibility is considered that chlorambucil interferes with histone gene expression at posttranscriptional or posttranslational levels. We also conclude that the inhibition of DNA synthesis by chlorambucil is most probably an indirect effect, a result of the inhibition of cells in the G2 phase of the cell cycle.

Expression of cell cycle regulatory factors in differentiating osteoblasts: postproliferative up-regulation of cyclins B and E.

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarial osteoblasts. Three developmental stages were examined: (a) proliferation; (b) monolayer confluency; and (c) mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and D1 persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor beta for 48 h during the proliferation period renders the cells incompetent for differentiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of ROS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cytoplasmic cyclin B when compared to the proliferation period. This observation is consistent with abrogation of both growth control and differentiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation.

Nuclear structure-gene expression interrelationships: implications for aberrant gene expression in cancer.

There is long-standing recognition that transformed and tumor cells exhibit striking alterations in nuclear morphology as well as in the representation and intranuclear distribution of nucleic acids and regulatory factors. Parameters of nuclear structure support cell growth and phenotypic properties of cells by facilitating the organization of genes, replication and transcription sites, chromatin remodeling complexes, transcripts, and regulatory factors in structurally and functionally definable subnuclear domains within the three-dimensional context of nuclear architecture. The emerging evidence for functional interrelationships of nuclear structure and gene expression is consistent with linkage of tumor-related modifications in nuclear organization to compromised gene regulation during the onset and progression of cancer.

A nuclear matrix antigen HeLa and other human malignant cells.

Antisera were obtained in rabbits to preparations of dehistonized chromatin from HeLa cells. By complement fixation assays, the antisera reacted with HeLa cell chromatin but only marginally with human placenta chromatin. The complement-fixing reactivity of the antisera was inversely related to the amount of dehistonized chromatin used for immunization. Immunochemical staining of electrophoretically separated chromosomal proteins transferred to nitrocellulose sheets revealed numerous antigens in chromatin preparations from several human tumors, placenta, and normal kidney. While immunoabsorption of the antisera with placenta chromatin removed some of the immunochemical staining, many of the electrophoretically separated antigens resisted repeated immunoabsorptions. However, further comparisons revealed that only one major protein antigen (band at an approximate molecular weight of 81,000) was represented in all the assayed human tumors while being absent from human placenta or kidney. Fractionation of HeLa cells into three cytoplasmic and several nuclear fractions showed that almost all the antigens recognized by antisera to dehistonized chromatin were nuclear. The antigenic protein with an approximately molecular weight of 81,000 was found associated with the nuclear matrix fraction.

Concerted control of multiple histone promoter factors during cell density inhibition of proliferation in osteosarcoma cells: reciprocal regulation of cell cycle-controlled and bone-related genes.

Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters. The results show that histone promoter binding activities HiNF-D, HiNF-P/H4TF-2, H4UA-1, and OCT-1, as well as AP-1 activity, are proliferation dependent. These factors decline coordinately during the cessation of proliferation in both ROS 17/2.8 bone tumor cells and normal diploid osteoblasts. Collective down-regulation of these trans-activating factors occurs in both cell types within the physiological context of constitutive regulation of ubiquitous transcription factors (Sp1, ATF, and CCAAT binding proteins). In addition, during growth inhibition of ROS 17/2.8 cells we observe a complex series of modifications in protein/DNA interactions of the osteocalcin gene. These modifications include both increased and decreased representation of promoter factor complexes occurring at steroid hormone response elements as well as tissue-specific basal promoter sequences. These results demonstrate cell growth regulation of the promoter factors binding to the proliferation-specific histone and tissue-specific osteocalcin genes during the cessation of proliferation.

Role of nonhistone chromosomal proteins in the regulation of histone gene expression.

Histone gene expression was studied during the cell cycle of continuously dividing HeLa S3-cells and following stimulation of confluent monolayers of WI-38 human diploid fibroblasts to proliferate. The presence of histone messenger RNA (mRNA) sequences was assayed by hybridization to a 3H-labeled single-stranded DNA complementary to histone mRNA's. In HeLa S3-cells, histone mRNA sequences were found in the nucleus and associated with polyribosomes during S phase but not during G1. Transcripts of S-phase chromatin contained histone mRNA sequences but those of G1 chromatin did not. Similarly, in WI-38 cells association of histone mRNA sequences with polyribosomes and transcription of histone mRNA sequences from chromatin parallel DNA replication. Together these results suggest that the regulation of histone gene expression resides, at least in part, at the transcriptional level. Chromatin reconstitution studies provide evidence that nonhistone chromosomal proteins play a key role in activation of histone gene transcription during the period of the cell cycle when DNA is replicated. Phosphate groups associated with the S-phase nonhistone chromosomal proteins appear to be functionally involved in the control of histone gene readout. Although WI-38 human diploid fibroblasts transformed by SV40 exhibit morphological and biochemical modifications characteristic of neoplastic cells, transcription of histone mRNA sequences remains unaltered.