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Results for “Histone Modifications”

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PubMed2025

CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

Humans↗
PubMed1998

Covalent modifications of histones: expression from chromatin templates.

Recent advances highlight the involvement of histone acetyltransferases in transcriptional activation and histone deacetylases in transcriptional repression. Transcription factors loaded onto regulatory DNA elements may recruit either coactivators with histone acetyltransferase activity or corepressors associated with histone deacetylases. The recruited enzymes may either acetylate or deacetylate proximal nucleosomal histones or nonhistone chromosomal proteins.

Acetylation↗
PubMed1975

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.

Animals↗
PubMed2003

Effects of polyamines on histone polymerization.

It is generally accepted that the nucleosome structure is not static, and that alternative conformations are adopted in response to several stimuli associated with the different functions. Histones are substrates for transglutaminase (TGase), and polymerized histone and polyamine binding histone have been suggested to play important roles in nucleus. We examined whether histone polymerization catalyzed by TGase was influenced by polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM). PUT inhibited histone polymerization, and SPD slightly prevented it. However, SPM slightly enhanced histone polymerization. These results indicate that the nuclear accumulation of the polyamines may play an important role in nuclear remodeling by histone modification. We speculate that histone cross-linking by TGase may be involved in the chromatin structure. Also, we propose that histone cross-linking by TGase may be responsible for the changes in DNA function such as transcription and replication and that TGase may be involved in cell growth and differentiation.

Animals↗
PubMed1970

Modification of histones during spermiogenesis in trout: a molecular mechanism for altering histone binding to DNA.

At a late stage of spermatogenesis in rainbow-trout testis, the entire complement of histones is replaced by newly synthesized protamine and histones are extensively phosphorylated and acetylated. Tryptic digestion of purified histones labeled by incubation of testicular cells with [(32)P]phosphate shows that phosphorylation occurs at a small number of seryl residues. Histone I (lysine-rich) is phosphorylated in the sequence Lys-Ser(PO(4))-Pro-Lys, which is located in the lysine-rich C-terminal region of the molecule. Histones IIb(1) (slightly lysine-rich) and IV (glycine, arginine-rich) give rise to the same phosphopeptide, Ac-Ser(PO(4))-Gly-Arg, which comprises the amino terminus of each histone. Thermolysin digests of phosphohistones IIb(1) and IV also released a phosphopeptide with composition corresponding to the first six residues of histone IV: Ac-Ser(PO(4))-Gly-Arg-Gly-Lys-Gly. An alpha-helical model of the N-terminal region of histone IV shows that this region is a possible DNA-binding site. Phosphorylation at serine 1 together with epsilon-amino acetylation at lysines 5, 8, 12, and 16 (observed in histone IV from trout testis) could profoundly modify ionic interactions and lead to an "unzipping" of histone IV from DNA

Animals↗
PubMed1972

Synthesis, acetylation, and phosphorylation of histone IV and its binding to DNA during spermatogenesis in trout.

During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [(3)H]aminoacids and inorganic [(32)P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [(3)H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [(3)H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total (3)H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an "obligatory" acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that epsilon-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH(2)-terminal region of histone IV to assume the correct conformation (in this case, an alpha-helix), and fit into the major groove of DNA. Deacetylation then "locks" histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known.

Acetates↗
PubMed1984

DNA repair-associated ADP-ribosylation in vivo. Modification of histone H1 differs from that of the principal acceptor proteins.

ADP-ribosylation in vivo of histone H1 was studied in hepatoma cells (Yoshida AH 7974) after treatment with the alkylating agent dimethyl sulfate for 30 min and compared with that of other polypeptides. In unstimulated cells, histone H1 was only a minor acceptor (less than 4%) of total monomeric and polymeric ADP-ribosyl residues. Induction of DNA repair by dimethyl sulfate treatment increased total mono(ADP-ribosyl) protein conjugates 1.6-fold whereas histone H1-linked mono(ADP-ribosyl) groups were elevated greater than 30-fold, thus accounting for nearly one-fourth of the net increase in monomeric ADP-ribosyl residues. In contrast, histone H1-associated poly(ADP-ribosyl) residues comprised only 2% of the total increase in poly(ADP-ribose). The extent to which the histone H1 population became ADP-ribosylated was low even in dimethyl sulfate-treated cells. Less than 2% of the histone H1 molecules were mono(ADP-ribosyl)ated and only 0.003% carried poly(ADP-ribosyl) chains when an average chain length of 10 is assumed. The principal polypeptide acceptors of alkylation-induced ADP-ribosylation were concentrated in two peaks, one migrating close to the position of core histones H3/H2B and accepting most of the induced mono(ADP-ribosyl) and poly(ADP-ribosyl) residues. The other (Mr = 110,000-160,000) resembled auto-modified poly(ADP-ribose) polymerase. Our data demonstrate marked differences of alkylation-induced (ADP-ribosyl)n protein patterns to analyses performed in vitro.

Adenosine Diphosphate Ribose↗
PubMed2001

Biochemical analysis of chromatin containing recombinant Drosophila core histones.

To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones. To this end, we synthesized the Drosophila core histones in Escherichia coli. The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues. The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP. With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones. We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones. In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template. Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications.

Animals↗