Posts Tagged ‘process’

‘K-to-M’ Histone Mutations: How Repressing Repressors May Drive Tissue-Specific Cancers

In 2012, investigators from multiple research institutions studying the sequence of the genome from cancer patients rocked the “chromatin world” when they independently reported that mutations in the gene that encodes histone H3.3 occurred in aggressive pediatric brain tumors. This finding was stunning, as researchers had never before associated histone mutations with any disease, much less a deadly tumor. What followed was a race by cancer researchers worldwide to discover how histone mutations might promote tumorigenesis.

Now a paper from a laboratory at the Stowers Institute of Medical Research reports the first animal model created to assess the molecular effects of two different histone H3.3 mutations in the fruit fly Drosophila. The study from a team led by Investigator Ali Shilatifard, Ph.D. published in the August 29, 2014 issue of Science, strongly suggests that these mutations actually could drive cancer and identifies interacting partners and pathways that could be targeted for the treatment of cancer.

Molecular biologists categorize these mutations as “K-to-M,” because a normal lysine residue (symbolized by K) in the protein is replaced by methionine (M) through mutations in the DNA sequence. In pediatric tumors, K-to-M mutations occurred at lysine residue 27 (K27) of histone H3.3. Researchers suggested that the presence of even a small population of these damaged proteins in the nucleus muffled a large repressor complex called PRC2. Normally, PRC2 acts as an enzyme to decorate histone lysines with one or more methyl groups, which silences gene expression by squeezing associated DNA into an impenetrable coil.

“Previously scientists knew that mutations in methylating enzymes like PRC2 occur in some cancers,” says Shilatifard. “What was surprising here was finding that mutation in one of the copies of the histone H3 gene, one of the proteins that PRC2 modifies, is associated with cancer. To figure out how that happened, we were interested in developing an in vivo model for the process in systems that we can study.”

The team first engineered a version of histone H3.3 that mimicked the K27-to-M mutation and then inserted that construct into embryonic fly tissues to produce the damaged protein in a living fruit fly. Using antibodies that recognize methylated lysines, they discovered that a dose of the mutant protein was sufficient to decrease global methylation of normal histone H3.3 proteins at K27, just as loss of the PRC2 repressor would. When the group engineered a similar K-to-M mutant at lysine 9 (K9), they saw similar results. This analysis of the H3K27 and H3K9 mutants confirmed in vivo that K-to-M mutations in histone H3.3 repress a key repressor, PRC2, but did not nail down how this happened.

“One question was whether a single amino acid change like this could alter the way histone H3.3 interacts with other proteins,” says Marc Morgan, Ph.D., a co-first author of the paper, “The mutant could be either losing or gaining something.” To determine which, the group collaborated with the Stowers Proteomics Center to compare factors binding to normal histone H3.3 versus the K-to-M mutants using mass spectrometry.

That analysis revealed that the presence of mutant histones globally dampens histone interactions with some of the usual repressor suspects. But in what Morgan calls an “Aha!” moment, they detected promiscuous association of a demethylase called KDM3B with the histone H3K9 mutant. “This suggests that these mutations inappropriately pull a demethylating enzyme onto chromatin, which then erases methylation marks in histones around it,” Morgan says.

Loss of methylation marks could allow expression of nearby genes. To confirm this, the group employed a Drosophila staining trick that allows experimenters to visualize how repressed genes are affected in entire tissues. The expression of KDM3B demethylase derepressed the gene expression in tissues such as salivary glands, just like the expression of the H3K9 mutant. This supports the idea that K-to-M mutations recruit a demethylase (like KDM3B) to demethylate chromatin on the K9 residue of H3.3 proteins in the neighborhood, where it likely uncoils chromatin to allow activation of genes that should be silenced.

This outcome could cause cancer in numerous ways. “One possibility might be that oncogenes that are usually silenced by methylation of residue 9 might be derepressed in the presence of the mutation,” says Hans-Martin Herz, Ph.D., a co-first author of the paper. But Herz is cautious in interpreting these findings, simply because, unlike the K27 mutations, mutations at residue K9 are not yet reported to be associated with cancer.

Intriguingly, other researchers recently reported a different K-to-M mutation (at residue 36 of histone H3.3) in chondroblastoma, a bone cancer sub-type. Why K-to-M mutations are so specific to a particular cancer is unknown, but Shilatifard says there can be little doubt that they play a central rather than a bystander role in tumorigenesis. “Uncharacterized K-to-M mutations may occur in other cancers,” he says. “Our work allows us to identify the molecular players involved in chromatin signaling in Drosophila and then apply those findings to human cells.”

source :

New tool aids stem cell engineering for medical research

“This free platform has a broad range of uses for all types of cell-based investigations and can potentially offer help to people working on all types of cancer,” says Hu Li, Ph.D., investigator in the Mayo Clinic Center for Individualized Medicine and Department of Molecular Pharmacology & Experimental Therapeutics, and co-lead investigator in the two works. “CellNet will indicate how closely an engineered cell resembles the real counterpart and even suggests ways to adjust the engineering.”

The network biology platform contains data on a wide range of cells and details on what is known about those cell types. Researchers say the platform can be applied to almost any study and allows users to refine the engineering process. In the long term, it should provide a reliable short cut to the early phases of drug development, individualized cancer therapies, and pharmacogenetics.

CellNet uses 21 cell types and tissues and data from 56 published human and mouse engineering studies as a basis for analyzing and predicting cell fate and corresponding engineering strategies. The platform also offers classification scores to determine differentiation and conversion of induced pluripotent stem cells. It reveals incomplete conversion of engineered microphages and hepatocytes. CellNet can be used for interrogation of cell fate following expression profiling, by classifying input by cell type, quantifying gene regulatory network status, and identifying aberrant regulators affecting the engineering process. All this is valuable in predicting success of engraftment of cancer tumors in mouse avatars for cancer and drug development research. CellNet can be accessed at

source :

How premalignant cells can sense oncogenesis, halt growth

Since the 1980s, scientists have known that mutations in a human gene called RAS are capable of setting cells on a path to cancer. Today, a team at Cold Spring Harbor Laboratory (CSHL) publishes experiments showing how cells can respond to an activated RAS gene by entering a quiescent state, called senescence.

CSHL Professor Nicholas Tonks and Benoit Boivin, now a University of Montreal Assistant Professor, co-led a team that traced the process in exquisite detail. They began by confirming that activation of mutant, oncogenic H-RAS, one of the human RAS oncogene variants, spurs cells to generate hydrogen peroxide (H2O2), a form of reactive oxygen species, or ROS. “Most people, when they think about ROS, think about the great damage they can do at high concentrations,” says Tonks. “But this research exemplifies how the controlled production of ROS in cells can play a beneficial role.”

The team showed how the production of ROS in response to oncogenic H-RAS enables cells to fine-tune signaling pathways, leading them to enter a senescent state. A key part of this process is the impact of ROS on a protein called PTP1B. Tonks discovered PTP1B some 25 years ago. It is an enzyme — one in a family of protein tyrosine phosphatases (PTPs), of which there are 105 in humans — that performs the essential biochemical task of removing phosphate groups from amino acids called tyrosines in other proteins. Adding and removing phosphates is one of the principal means by which signals are sent among proteins.

In cells with oncogenic H-RAS, ROS is produced in small quantities, sufficient to render PTP1B inactive. The team found that with the phosphate-removing enzyme unable to do its usual job, a key protein called AGO2 remains phosphorylated — with the consequence that it can no longer do what it normally does, which is engage the cell’s RNA interference machinery. In normal cells, the RNAi machinery represses a gene called p21. But in this specific condition — with H-RAS oncogenically activated, PTP1B inactivated by ROS, and RNAi disabled — p21 proteins begin to accumulate unnaturally, the team discovered.

“This is the key step — accumulation of p21 proteins effectively halts the cell cycle and enables the cell to enter the senescent state,” explains Ming Yang, a doctoral student in the Tonks lab. She and Astrid Haase, Ph.D., a postdoctoral investigator in the laboratory of CSHL Professor Greg Hannon, are the first two authors, respectively, on the team’s paper, published in Molecular Cell.

“This is confirmation of a hypothesis we presented five years ago,” Tonks says. “We knew that oncogenic RAS induced the production of ROS. We proposed that this would lead to regulation of PTPs, and using the example of PTP1B this is precisely what the team discovered in this work — showing also how inactivation of this PTP is part of a complex signaling cascade that can culminate in the induction of senescence.”

ROS have been linked to the pathogenesis of several diseases including Alzheimer’s, diabetes and heart failure. “By showing that PTP1B inactivation by oxidation prevents AGO2 from doing its job, we make a clear link between ROS and gene silencing which could also be observed in other pathologies” says Boivin. Hence, the role of PTP1B in keeping the RNAi machinery active could have important ramifications.

Entering senescence is not enough to arrest oncogenesis completely. Oncogenic mutations typically multiply as cancers evolve to promote their survival and proliferation. But the current work does show the potential importance of knowing the genetic background of a cancer patient, for there are windows of time — narrow though they may be — in which naturally occurring processes induce pauses in growth.

source :