Histone Methylation Profiles in Autism

August 30, 2011

The effects of autism spectrum disorders (ASD) are mostly seen in the brain, so a new study published by researchers from the University of Massachusetts Medical School focused on mapping the H3K4me3 epigenomes in prefrontal cortex (PFC) neurons of autistic individuals to better understand the complex condition.

The project compared autistic PFC neuron profiles with a panel of controls from subjects ranging in age from infancy to 70 years. The UMass team used an H3K4me3 antibody in ChIP-Seq to generate genome-wide maps of the H3K4me3 mark in neuronal nuclei from the PFCs of 16 subjects diagnosed with ASD, plus control samples. Here are some of the findings from the research analysis:

  • Autistic patients didn’t show a global difference in H3K4me3 occupancy at known promoters, and also had an age-appropriate promoter H3K4me3 profile.
  • Neuronal chromatin from some autism cases, however, exhibited excessive spreading of H3K4me3 marks into nucleosomes farther away from the transcription start sites.
  • Location analysis found that 503 H3K4me3 loci were increased (autism-up) and 208 loci were decreased (autism-down) in a subset of autistic subjects. Of those, 330 autism-up loci and 139 autism-down loci overlapped with promoters.

Based on their research findings, the scientists concluded that certain autistic individuals are affected by a loss or excess of H3K4me3 at hundreds of loci, leading to dysregulated transcript expression and traits associated with ASD. These changes are highly variable between patients and indicate complex interactions between the genetic and epigenetic mechanisms affecting autism spectrum disorders.

See the details in Archives of General Psychiatry, November 2011.

Enhancing Chromatin Control of ES Cell Pluripotency

August 30, 2011

One of the most defining characteristics of cells like embryonic stem cells (ESCs) is pluripotency, or the ability to differentiate into any cell type. This ability intrigues researchers who hope to harness that potential for a wide range of therapeutic applications.

Recent evidence has implicated epigenetic regulation of ESC pluripotency, so scientists from UC San Diego set out to explore in more depth the role that chromatin states play during stem cell differentiation.

The UCSD team ran genome-scale ChIP-Seq and ChIP-chip experiments on ES cells before and after differentiation, looking for changes in chromatin states, especially at promoter and enhancer regions.

Here's what they found:
• Many promoters associated with pluripotency- or cell fate–related genes had switched chromatin states from methylation to acetylation at H3K27 marks, sure signs of a change in gene expression.
• A majority of enhancers investigated either gained or lost H3K4me1 or H3K27ac modifications during differentiation.
• The enhancer changes correlated to cell type–specific patterns in CTCF-organized regulatory domains (CORDs), indicating a potential regulatory network.
• A certain group of enhancers, marked by H3K4me1 that then becomes acetylated at differentiation, were found to be poised enhancers critical to kicking off the differentiation process.

All together the researchers found new evidence that epigenetic mechanisms, including chromatin states, regulate pluripotency in ES cells.

For more details, see the full article in Cell Research, August 2011.

Does 5hmC Help Cancer Get Stemmy?

September 30, 2011

There's quite a bit of speculation on the significance of 5-hydroxymethylcytosine (5hmC) in regulating gene expression, yet the actual function of 5hmC has yet to be determined. A study mapping the distribution of this epigenetic modification may shed some light on its role in differentiation and carcinogenesis, and will certainly supply fodder for further research and discussion.

Srinivasan Yegnasubramanian and his team at Johns Hopkins found that across a range of embryonic and adult tissue, cells that were more stem- and progenitor-like had greatly reduced staining for the 5hmC modification when compared with more differentiated cells.

Similarly, tumor cells—which have presumably become de-differentiated—evinced significantly less 5hmC than their normal counterparts.

Their other key findings:

  • In hierarchically arranged tissue like the colon, cervix, oral mucosa, and bladder, more differentiated luminal/apical cells showed greater evidence of 5hmC modification than did the more regenerative basal cells.
  • Similarly, the CD34– bone marrow–derived hematopoietic cell compartment, where more mature blood cells are found, exhibited much higher 5hmC content than did the CD34+ compartment representing stem and progenitor cells. These findings may indicate a role of 5hmC in differentiation.
  • There was a profound reduction in 5hmC staining in breast, colon, and prostate cancer cells when compared to adjacent normal cells in the same samples. This was not associated with either grade or stage, suggesting that global loss of 5hmC may be an early event in carcinogenesis.

For more findings and the immunohistochemical staining technique that made it all possible, see Oncotarget, August 2011.

Stem Cells Get with the MicroRNA Program

May 15, 2011

As mounting evidence shows that microRNAs (miRNAs) are critical to running posttranscriptional genetic programs in stem and progenitor cells, researchers from Stanford University decided to take an in-depth look at what miRNA expression profiles could tell us about these programs.

Using TaqMan® MicroRNA Assays, the team poured over miRNA expression data in the stem cells and their differentiated brethren from multiple adult tissues.

Their work noteworthy features, including:

  • Certain signatures are unique to blood, muscle, and neural stem cell populations
  • Some miRNA patterns mark the transition from stem cells to differentiating progenitor cells
  • Stem/progenitor transition miRNAs (SPT-miRNAs) predict the effects of genetic alterations, like the loss of PTEN on self-renewal and proliferation potentials of mutant stem cells
  • SPT-miRNAs control the self-renewal of embryonic stem cells and hematopoietic stem cells (HSCs)
  • SPT-miRNAs were also found to regulate genes that are known to be involved in HSC self-renewal (HOXB6 and HOXA4)

By using their analysis to map out the miRNA programs behind key processes in normal and aberrant stem and progenitor cell development, the Stanford authors hope to create a new foundation for dissecting the posttranscriptional networks in stem cells.

For more details, see the publication in Genome Research, May 2011

TaqMan® MicroRNA Assays are For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

DNA Methylation Helps Keep Tabs on Hepatogenesis

May 4, 2011

In Vitro hepatogenesis is a slick model system for studying liver development, not to mention that hepatocytes created from human embryonic stem cells (hESCs) are great for drug testing and have the potential to treat liver diseases as well. To get a better understanding of this system, Korean scientists profiled gene expression and DNA methylation at three points along the way to in vitro hepatogenesis–hESCs, definitive endoderm (DE), and hepatocytes.

The group from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) started off using microarrays to identify 525 state-specific expressed genes, 67 of which showed significant negative correlation between gene expression and DNA methylation.

The team next wanted to scan genome-scale methylation changes outside of the promoter regions, so they employed high-throughput sequencing (MBD-seq) of the methylated DNA captured by the MBD2 protein in the Invitrogen™ MethylMiner™ Methylated DNA Enrichment Kit. They found many dynamic intergenic methylation changes going on during differentiation, including:

  • 16 ESC-specific methylation markers, seven of which are newly identified
  • 11 DE methylation markers, including 3 that were previously unknown
  • 40 hepatocyte-specific methylation markers, most of which (31) were previously unknown

Together, these genetic markers can be powerful research tools for the study of in vitro hepatogenesis and liver development.

In addition to uncovering genes that have lineage-specific expression, are demethylated during hepatogenesis, and show dynamic intergenic DNA methylation changes throughout the genome during hESC differentiation, the authors hope their work will help to clarify the mechanisms of hESC differentiation and to gain a better understanding of liver disease.

For more information, see the publication in Human Molecular Genetics, May 2011

The MethylMiner™ Methylated DNA Enrichment Kit is for Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Cancer Cells Display Different Methylomes

New research finds that tumor cells don't all have the same methylation patterns. In fact, variation within tumors gives cells an advantage in challenging environments, and even helps them avoid detection and treatment.

Recently, the Feinberg lab at Johns Hopkins University ran whole-genome bisulfite sequencing experiments on Applied Biosystems® SOLiD® sequencers to study the DNA methylation differences between cancer types and normal cells. Here's what their analysis revealed:

  • Increased DNA methylation variation in several cancer types compared to normal tissues
  • Large regions (5 kb to 10 Mb) of DNA hypomethylation that cover half the genome
  • Small, differentially methylated regions (DMRs) that seem to be involved in the loss of methylation boundaries

The team believes that their experiments have uncovered two new attributes of cancer. The first is a loss of stability of normal boundaries of methylation around CpG islands. And the second is a large-scale hypomethylation of genomic regions.

This epigenetic instability allows cancers to quickly adapt and survive in a changing environment, and stochastic variation in methylation enhances that survival. The same sites of stochastic variation are also critical for normal development. As the data indicate, the most variable sites in colon cancer are also the most variable in other common cancers, and this variation makes it possible to discriminate between cancer and normal tissues.

These mechanisms also could explain the heterogeneity so often found within tumors, and might have implications in the future diagnosis and treatment of tumors.

The SOLiD® sequencer is for research use only, and is not intended for animal or human therapeutic or diagnostic use.

Find the full paper in Nature Genetics, June 2011

Sequencing Identifies IsomiRs in Pre-eclampsia

June 22, 2011

As next-generation sequencing becomes more accessible, researchers are finding out that nucleic acids are a lot more complex than they ever imagined. For instance, miRNA genes can create multiple variants, called isomiRs, just like protein-coding genes. New research using advanced sequencing probed the differences in the isomiRs from women with pre-eclampsia, a serious pregnancy condition characterized by high blood pressure and increased protein in the urine.

Using the SOLiD® sequencing platform from Life Technologies, the Chinese researchers looked at isomiRs in placental samples from healthy patients compared to others with mild or severe pre-eclampsia. Their data showed that:

  • In every group, several isomiRs for each miRNA gene existed. The most common variants had 3' deletions, making them shorter than the standard 22-nt miRNA.
  • Over 30% of isomiRs had additional nucleotides at their 3' ends, usually a single adenosine. IsomiRs with 3' additions might be shorter, longer, or the same length as the typical 22-nt miRNA.
  • Relative to normal miRNAs, the 3' variants were expressed at low levels in all three groups. However, compared with the pre-eclampsia groups, normal samples had higher expression of isomiRs with 3' additions.
  • isomiR differences were found between normal and disease samples, as well as between mild and severe pre-eclampsia samples.

What's with all the variation? Researchers aren't sure why extra nucleotides are being added to the 3' ends of miRNAs, but they propose that the 3' addition might alter miRNA stability or function.

The SOLiD® sequencer is for research use only, and is not intended for animal or human therapeutic or diagnostic use.

Read the full article in PLoS One, June 2011