scholarly journals The Forkhead Transcription Factor FoxI1 Remains Bound to Condensed Mitotic Chromosomes and Stably Remodels Chromatin Structure

2006 ◽  
Vol 26 (1) ◽  
pp. 155-168 ◽  
Author(s):  
Jizhou Yan ◽  
Lisha Xu ◽  
Gregory Crawford ◽  
Zenfeng Wang ◽  
Shawn M. Burgess
Keyword(s):  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Dnase I ◽  
Higher Order ◽  
Micrococcal Nuclease ◽  
Stable Cell Line ◽  
Order Structure ◽  
Dna Fragments ◽  
Mitotic Chromosomes

ABSTRACT All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure

1989 ◽  
Vol 9 (7) ◽  
pp. 3136-3142
Author(s):  
U Maschek ◽  
W Pülm ◽  
S Segal ◽  
G J Hämmerling
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Chromatin Structure ◽  
Dnase I ◽  
Class I ◽  
Micrococcal Nuclease ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.

scholarly journals Higher-order structure of human mitotic chromosomes.

1977 ◽  
Vol 74 (4) ◽  
pp. 1595-1599 ◽  
Author(s):  
A. L. Bak ◽  
J. Zeuthen ◽  
F. H. Crick
Keyword(s):  
Higher Order ◽  
Order Structure ◽  
Mitotic Chromosomes

scholarly journals Regulation of the higher-order structure of chromatin by histones H1 and H5.

1981 ◽  
Vol 90 (2) ◽  
pp. 279-288 ◽  
Author(s):  
J Allan ◽  
G J Cowling ◽  
N Harborne ◽  
P Cattini ◽  
R Craigie ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Single Species ◽  
Histone H1 ◽  
Higher Order ◽  
Linker Histone ◽  
Micrococcal Nuclease ◽  
Order Structure ◽  
Chicken Erythrocyte ◽  
Base Pairs ◽  
Native Chicken

Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.

scholarly journals Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

2008 ◽  
Vol 183 (7) ◽  
pp. 1193-1202 ◽  
Author(s):  
Owen J. Marshall ◽  
Alan T. Marshall ◽  
K.H. Andy Choo
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Protein A ◽  
Three Dimensional ◽  
Higher Order ◽  
Order Structure ◽  
Mitotic Chromosomes ◽  
Centromeric Chromatin ◽  
Centromere Protein A ◽  
Transmission Electron ◽  
Histone H3 Variant

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.

scholarly journals Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure.

1989 ◽  
Vol 9 (7) ◽  
pp. 3136-3142 ◽  
Author(s):  
U Maschek ◽  
W Pülm ◽  
S Segal ◽  
G J Hämmerling
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Chromatin Structure ◽  
Dnase I ◽  
Class I ◽  
Micrococcal Nuclease ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.

scholarly journals Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of theHPRT Promoter

2001 ◽  
Vol 21 (22) ◽  
pp. 7682-7695 ◽  
Author(s):  
Chien Chen ◽  
Thomas P. Yang
Keyword(s):  
Chromatin Structure ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Minimal Promoter ◽  
Micrococcal Nuclease ◽  
Promoter Regions ◽  
Permeabilized Cells ◽  
Active Allele

ABSTRACT Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Higher order structure of mitotic chromosomes

1978 ◽  
Vol 283 (997) ◽  
pp. 415-416 ◽  
Keyword(s):  
Human Fibroblasts ◽  
Higher Order ◽  
Order Structure ◽  
Mitotic Chromosomes ◽  
Cylindrical Structure

From observations on the partial disintegration of mitotic chromosomes isolated from human fibroblasts we propose that human mitotic chromatids are characterized by a rather simple organization based on the folding and coiling of a long, regular, hollow cylindrical structure the unit fibre , with a diameter of about 400 nm (Bak, Zeuthen & Crick 1977). This structure is postulated to consist of a super-solenoid formed by the coiling of the 30 nm solenoid (itself formed by coiling of the string of nucleosomes).

scholarly journals Chromatin remodelling of the cardiac β-myosin heavy chain gene

1998 ◽  
Vol 330 (2) ◽  
pp. 871-876 ◽  
Author(s):  
Weei-Yuarn HUANG ◽  
Choong-Chin LIEW
Keyword(s):  
Gene Expression ◽  
Chromatin Structure ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Proximal Promoter ◽  
Chain Gene ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Hamster Heart ◽  
Adult Hamster

To investigate the role of chromatin structure in cardiac gene expression, we used the DNase I and micrococcal nuclease to probe the chromatin structure of the hamster cardiac β-MyHC gene. Two cardiac-specific DNase I hypersensitive sites (DHS) were identified, one of which was mapped to the -2.3 kb (β-2.3 kb) region and the other to the proximal promoter region of the β-MyHC gene. The two sites were readily detectable using nuclei from neonatal hamster heart; however, the proximal promoter site disappeared when adult hamster heart nuclei were used, and the -2.3 kb site decreased in intensity. We were able to demonstrate the gradual disappearance of this proximal promoter DHS by comparing heart nuclei isolated from animals at late-gestation and 1-day-old stages. Furthermore, injecting thyroid hormone caused the disappearance of the proximal promoter DHS in late gestational fetal ventricular nuclei. Digestion of nuclei from various tissues by micrococcal nuclease revealed that the β-MyHC gene proximal promoter exists in an array of three specifically-positioned nucleosomes only in fetal heart chromatin. The β-MyHC gene proximal promoter is DNase I hypersensitive within one of the nucleosomal particles. Our data suggest that chromatin structure may participate actively in cardiac gene expression.

Sign in / Sign up

Export Citation Format

Share Document