H3 Incorporation Research Articles

Dynamic Histone H3 Incorporation Fuels Metastatic Progression

Treating metastatic cancers is an ongoing challenge in oncology. A recent paper by Gomes and colleagues proposes histone H3 variant dynamics as major regulator of cell fate transition during metastasis and suggests histone chaperones as therapeutic targets for invasive carcinoma.

Histone H3 Dynamics Reveal Domains with Distinct Proliferation Potential in the Arabidopsis Root.

A coordinated transition from cell proliferation to differentiation is crucial for organogenesis. We found that extensive chromatin reorganization, shown here for histone H3 proteins, characterizes cell population dynamics in the root developmental compartments. The canonical H3.1 protein, incorporated during S-phase, is maintained at high levels in cells dividing at a high rate but is massively evicted in cells undergoing their last cell cycle before exit to differentiation. A similar pattern was observed in the quadruple mutant for the H3.1-encoding genes HTR1, HTR2, HTR3, and HTR9 (htr1,2,3,9), in which H3.1 is expressed only from the HTR13 gene. H3 eviction is a fast process occurring within the G2 phase of the last cell cycle, which is longer than G2 in earlier cell cycles. This longer G2 likely contributes to lower the H3.1/H3.3 ratio in cells leaving the root meristem. The high H3.1/H3.3 ratio and H3.1 eviction process also occurs in endocycling cells before differentiation, revealing a common principle of H3 eviction in the proliferating and endocycling domains of the root apex. Mutants in the H3.1 chaperone CAF-1 (fas1-4) maintain a pattern similar to that of wild-type roots. Our studies reveal that H3 incorporation and eviction dynamics identify cells with different cell division potential during organ patterning.

Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest.

In order to maintain a stable genome, cells need to detect and repair DNA damage before they complete the division cycle. To this end, cell cycle checkpoints prevent entry into the next cell cycle phase until the damage is fully repaired. Proper reentry into the cell cycle, known as checkpoint recovery, requires that a cell retains its original cell cycle state during the arrest. Here, we have identified Tousled-like kinase 2 (Tlk2) as an important regulator of recovery after DNA damage in G2. We show that Tlk2 regulates the Asf1A histone chaperone in response to DNA damage and that depletion of Asf1A also produces a recovery defect. Both Tlk2 and Asf1A are required to restore histone H3 incorporation into damaged chromatin. Failure to do so affects expression of pro-mitotic genes and compromises the cellular competence to recover from damage-induced cell cycle arrests. Our results demonstrate that Tlk2 promotes Asf1A function during the DNA damage response in G2 to allow for proper restoration of chromatin structure at the break site and subsequent recovery from the arrest.

Abstract LB-348: Chemoresistance in pancreatic cancer is conferred by MUC1 cytoplasmic tail binding to the promoter region of the ABCC1 (MRP-1) gene

Abstract Introduction: The high mortality rate of pancreatic cancer (PC) is in part, due to their high propensity for metastasis and multidrug resistance. The three most common ways by which cancer cells acquire chemoresistance is via deregulation of apoptotic pathways, upregulation of survival pathways and upregulation of multi-drug resistance (MDR) genes. The MDR genes encode for ATP-binding cassette (ABC) membrane transporters, which extrudes chemotherapeutic drugs from cancer cells. However, the mechanisms behind the up-regulation of the MDR genes are not entirely understood. More than 90% of metastatic PC patients overexpress MUC1 (CD227), a membrane tethered mucin glycoprotein that has been linked to increased metastatic phenotype and drug resistance. The cytoplasmic tail (CT) of MUC1 has seven conserved tyrosine residues which are important for the signal transduction role of MUC1. The aim of this study was to understand the underlying mechanism by which MUC1 induces drug resistance in PC cells. Method: We used a panel of PC cells lines that endogenously express low or high levels of MUC1. The cells lines expressing low endogenous MUC1 were stably infected with the retroviral vector containing empty plasmid (Neo), or plasmid carrying full-length MUC1 (MUC1 WT), or full length MUC1 with the tyrosine residues of MUC1 CT mutated to phenylalanine (MUC1 Y0). The cells were treated with three commonly used chemotherapeutic/preventive drugs (Gemcitabine, Etoposide and Celecoxib). The level of dead cells was evaluated by H3 incorporation assay and Annexin V staining. We also checked for the activation of prosurvival pathways Erk1/2 and PI3K by Western Blot. The mRNA level of MDR genes were determined by RTPCR. CHIP (Chromatin Immunoprecipitation) was performed to determine the localization of MUC1 CT to the promoter region of ABCC1 gene (MRP1). Results: Our results indicate that PC cells expressing MUC1 WT or MUC1 Y0 have intrinsic resistance to chemotherapeutic, compared to cells expressing low levels of MUC1. We observed an enhanced activation of the prosurvival pathways Erk1/2 and PI3K in MUC1 high PC cells, compared to cells expressing low MUC1 or MUC1 Y0. Upon inhibition of the PI3K/Akt pathway, a partial loss in the chemoresistance was seen, indicating that this pathway is partially responsible for the chemoresistance. DNA microarray and RT PCR showed that the expression of MDR genes (ABCC1, ABCC3, ABCC5 and ABCB1) was significantly higher in PC cells expressing MUC1 WT or MUC1 Y0. Most importantly, for the first time, we showed that MUC1 CT localizes to the promoter region of a MDR gene, ABCC1. This suggests that MUC1 directly drives the transcription and expression of ABCC1 gene. The implications of the data are clinically significant as small molecule inhibitors can be designed to specifically block MUC1 CT signaling and transcriptional activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-348. doi:1538-7445.AM2012-LB-348

The Histone Variant H3.3 Regulates Gene Expression during Lytic Infection with Herpes Simplex Virus Type 1

It has been proposed that incorporation of the histone variant H3.3 within actively transcribed regions of a genome helps to facilitate transcription. In this report we use lytic infection by herpes simplex virus type 1 (HSV-1) as a model to examine the temporal profile of histone H3 incorporation and to determine whether the variant histone H3.3 has a direct effect on transcription. We find that canonical H3.1 and variant H3.3 exhibit distinct temporal associations with the genome in cell lines expressing equal amounts of epitope-tagged H3 variants. At the earliest times examined after infection, the HSV-1 genome is incorporated into chromatin that predominantly contains the variant H3.3, whereas incorporation of canonical H3.1 occurs later in infection and is dependent on replication of the HSV-1 genome. Further, inhibition of H3.3 association, via reduced expression of the H3.3 chaperone HIRA, significantly reduces the levels of HSV-1 mRNA. These findings show that incorporation of H3.3 facilitates transcription, and they provide new evidence for a regulatory role of chromatin composition during HSV-1 acute infection.

Continuous Histone H2B and Transcription-Dependent Histone H3 Exchange in Yeast Cells outside of Replication

We investigated the dynamics of histone-DNA interactions in yeast by using inducible forms of epitope-tagged histones H2B and H3. Chromatin assembly of newly synthesized histones was assessed by chromatin immunoprecipitation in G1-arrested cells to prevent replication-coupled histone incorporation. We find that while histone deposition within a subtelomeric region is strictly linked to DNA replication, histone H2B is continuously incorporated at the promoter and coding regions of both transcriptionally active and inactive loci. In contrast, incorporation of histone H3 occurs only at active genes, being predominant at the promoter and showing a dynamics along the gene that inversely correlates with the average nucleosomal density. Similar results were obtained with N-terminally truncated H2B and H3 variants. We infer that replication-independent incorporation of H2B and H3 are distinct events, each occurring independently of the histone tail, and that nucleosome loss at active promoters reflects a dynamic equilibrium between histone deposition and dissociation.

Thymidine H3 incorporation in the nurse cells of amphigonic and parthenogenetic ovaries of Megoura viciae (Hom. Aph.).

In the amphigonic females ofM. viciae an active nuclear incorporation of thymidine H3 occurs during growth which may be attributed to continuous endomitotic divisions. However, even when the nurse cells are functioning fully, and when the nuclei appear to have achieved maximum development, incorporation of the thymidine H3 continues, and, as was seen in other insects, does not affect all the nuclei. Incorporation would therefore seem to be due not to the continuance of endomitotic divisions but rather to the synthesis of metabolic DNA. On the other hand, even if one supposes that in the nurse cells of the amphigonic ovary endomitotic processes continue right up to the end of vitellogenesis of the amphigonic winter egg, this is quite out of the question so far as the parthenogenetic ovary is concerned. Diploid nurse cells are functioning continuously, since in a parthenogenetic ovary a great many ovocytes reach maturity one after the other, passing through all stages of development to produce the embryos. The nurse cells always retain, in such cases, their characteristic appearance right from the beginning of their differentiation3 and therefore thymidine H3 incorporation cannot be ascribed to continuous endomitotic divisions. It can therefore be assumed that the active synthesis which occurs in the nuclei does not concern genetically stable DNA but a metabolic DNA. The above results thus add new weight to the assumption by former authors that metabolic DNA may be synthesized in the nurse cells of amphigonic insects as well.

H3 Incorporation into Ascites Tumour and Tissue Culture Cells Exposed to Synkavit and its Tritiated Analogue

H3 Incorporation into Ascites Tumour and Tissue Culture Cells Exposed to Synkavit and its Tritiated Analogue

H3 Incorporation into Ascites Tumour and Tissue Culture Cells Exposed to Synkavit and its Tritiated Analogue

Autoradiographic studies of living normal and malignant cells show a rapid incorporation of the tritium of the tetrasodium 2-methyl-1: 4-naphthaquinol (6-T) diphosphate into the cells. A selectivity of uptake by the tumor cells measured by grain counts was demonstrated. (auth)