Arches. Photo by Daniel Chia
Jun
30
2010

Histones and HDAC Inhibitors

Histone deacetylases (HDACs) are enzymes involved in expression of DNA.  Blocking these enzymes with HDAC inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) has beneficial effects on fruit-fly and mouse models of HD. Why are HDACs significant in the study of HD and how could they lead to HD therapies? To answer these questions, we will explore histones and their role in gene expression, learn about certain histone-modifying enzymes, and then address how regulation of these enzymes can have potentially beneficial effects for an individual with HD.

Histones and DNA^

If you were to stretch out all of the DNA in the human genome, it would span approximately 102 centimeters. This length is tremendous given that the average diameter of a mammalian cell’s nucleus, which is where DNA is stored, is only 5 micrometers. Given that the cell’s total length of DNA is over 20,000 times longer its storage space, fitting DNA into the nucleus requires packing it within a tightly-condensed state known as chromatin. In its chromatin form, DNA is organized in structures called nucleosomes, which consist of both DNA and proteins known as histones. Each nucleosome has 147 base pairs of DNA wrapped around a scaffolding of eight histones like twine around a spool. Thus, histones provide the structural support for packaging DNA.

Not only do histones provide a backbone for packaging DNA, they also regulate the function of DNA, which is to replicate the body’s cells and to transcribe all of the body’s protein building blocks. When the cell needs to divide or transcribe its genes, signaling proteins  are sent to interact with the “tail” of amino acids on the histones, causing them to unwind their DNA. When DNA is unwound, its distinct strands can be accessed by the cell’s replication or transcription machinery.

DNA accessibility can be regulated by histones by adding and then subsequently removing molecules such as acetyl groups, methyl groups, and phosphate groups to or from specific sites on the histone tail. These histone modifications are performed by enzymes that are specialized to attach or remove a specific group. For example, enzymes known as histone methyltransferases are responsible for attaching methyl groups to histones. These attached groups are small compared to the size of the structure they are attached to, but their presence or absence on the histone have a big effect on the accessibility of the DNA in its tightly-condensed chromatin form. For instance, adding a methyl group to the ninth lysine on histone 3 represses transcription, while adding it to the seventeenth arginine activates transcription. The significant effect of this methyl group and other histone modifiers has been attributed to two different mechanisms:

  1. The binding of modifiers disrupts contacts between nucleosomes, resulting in the unraveling of the DNA.
  2. The bound modifiers attract or repel other proteins that initiate a variety of enzymatic activities (e.g. prepare the DNA for transcription).

Clearly, it is very important for cells to precisely control which modifications go on which part of the histone. The huge variety of different combinations of attached groups has led scientists to suggest that these modifications serve as a “code” that tells the cell what to do with the DNA wound around the histone.

Histone acetylation and HD^

Because the chromatin structure is so important in regulating in the accessibility of DNA, histones and the enzymes that modify them play significant roles in controlling which genes are expressed and which are turned off. This method of regulation is very relevant in the nervous system, which depends on the coordinated expression of many different genes in order to develop and maintain itself. For example, recall that all neurons are descendants of embryonic stem cells, cellular progenitors that have the potential to turn into many other types of cells. What these stem cells ultimately become depends on the signals that they receive from their environment. (For more information on stem cells, click here.) One way that these signals may “tell” stem cells what to become is through histone modifications. This makes sense when you consider the importance of chromatin structure in determining the access of genes—turning different genes “on” and “off” results in stem cells taking on different fates. As we will soon see, in an organ as critical and complex as the brain, even small changes in gene expression can have big effects on function.

The addition of a chemical structure known as an acetyl group to histones is associated with activating transcription. This addition, which is known as acetylation, is performed by enzymes known as histone acetyltransferases (HATs) and produces a more transcriptionally-active form of chromatin. Conversely, the removal of attached acetyl groups by enzymes called histone deacetylases (HDACS) represses transcription. Varying the amounts of active HATs and HDACs allows the cell to control the accessibility of its chromatin, and thus which genes are turned on and when. One simple way to think about this relationship is a seesaw, with the left side signifying a certain gene is “on” and the right side signifying that a gene is “off.” If there are more HATs on the left side than there are HDACs on the other side, more histones will be acetylated than not and the seesaw will tilt towards the gene being transcribed. On the other hand, if HDACs outnumber HATs, acetyl groups will be removed faster than they are added and the transcription of the gene is repressed. Of course, the control of gene transcription is more complex than a seesaw of HDACs and HATs. However, this model provides a simple way of understanding what can go wrong if the balance between HDACs and HATs goes changes, as what appears to happen in HD.

Mutant huntingtin and acetylation: ^

An imbalance in histone modifications occurs in HD and researchers are trying to correct this problem as a therapeutic strategy. The mutated version of the huntingtin protein has been shown to disrupt transcription. (For more information about mutant huntingtin, click here.) Many genes important for the proper functioning of neurons, such as those responsible for neurotransmitter signaling, are down-regulated, or show reduced expression, in the brains of HD patients and animal models. The reduced output of these genes plays a major role in the massive neurodegeneration seen in HD and is likely at least partially due to mutant huntingtin’s effect on histones. Experiments examining the chromatin of genes known to be downregulated in both HD cell lines and HD mice and found that their histones contained fewer acetyl groups than usual. Recalling the well-established role that acetylation plays in transcriptional activation, these results provide a way of understanding how transcription is affected in HD.

Further investigations have provided more insight into what actually causes the reduced acetylation seen in HD. Mutant huntingtin has been observed to interact with both HATs, the enzymes that add acetyl groups, and HDACs, the enzymes that remove them. While there are some indications that mutant huntingtin may directly act on HATs to de-active them, there has been more evidence suggesting that the mutated protein affects the histone-modifying enyzmes by making them unavailable to do their jobs. This latter mechanism is a recurring one in HD—mutant huntingtin sequesters transcription factors and co-activators (molecules that help transcription factors bind to DNA), resulting in abnormal gene expression. But what happened to these coactivators and transcription factors? In order to answer this question, we need to review some of the disease mechanisms that take place in the cells with mutant huntingtin.

A Brief Review of HD Mechanisms

Fig N-3: NI Formation

The expanded CAG repeat found in the HD gene produces a mutant form of the huntingtin protein. At some point in the life of mutant huntingtin, enzymes known as caspases cleave the protein, producing huntingtin fragments. These fragments are transported into the nucleus of the HD brain cell, or neuron, where they aggregate to form neuronal inclusions (NIs) in the nucleus. Figure 3 shows a diagram depicting the formation of NIs from the altered huntingtin protein. (For more information about the mutant huntingtin protein and protein aggregation, click here.)

Fig N-4: Transcription and NIs

Recent studies showed that the NIs “trap” various co-activators and transcription factors and prevent them from doing their jobs. This discovery led scientists to speculate that one of the ways by which HD progresses is through the loss of transcription of several key genes essential for cell survival. It was then important for scientists to try to identify which molecules are being trapped by the NIs so that this molecular “trapping” can be counteracted.

One such molecule called CREB-binding protein (CBP), which is an HDAC, appears to associate with NIs. CBP is a co-activator because it induces histones to adopt a more open chromatin configuration, allowing transcription factors to bind.  If CBPs are trapped by the NIs, transcription factors can’t access DNA and certain genes are not transcribed.  Sequestering CBP in HD especially affects p53, a transcription factor that regulates neuronal death, but is better known for its role as a tumor-suppressor protein. When few CBP molecules are available to co-activate p53, it is unable to access and bind to DNA. Without p53, abnormal gene transcription and expression occurs, and scientists speculate that this error in transcription may lead to cell death.

Fig N-5: A Detailed Look at Transcription and NIs

In summary, the altered huntingtin protein forms NIs that trap coactivators such as CBP. Loss of CBP results in the loss of histone acetylation, which in turn results in the inability of the transcription factor p53 to bind to DNA. Drugs such as HDAC inhibitors that could compensate for the loss of the coactivator CBP could, in theory, be possible treatments for HD. The following section summarizes some of the recent studies that test this theory using animal models.

The disruption of localized processes, such as p53 binding, by mutant huntingtin results in global changes to the cell. Gene profiling studies, which examine the expression of a tremendous number of genes, have found significant differences in the messenger RNAs (mRNAs) that are produced in individuals with HD compared to those unaffected by the disease. Recall that mRNAs are transcribed from DNA and form the genetic code that is “read” to produce proteins. The changes seen in the expression profiles of individuals affected by HD suggest that the disease impacts transcription at a global level.

The disruption of localized processes, such as p53 binding, by mutant huntingtin results in global changes to the cell. Gene profiling studies, which examine the expression of a tremendous number of genes, have found significant differences in the messenger RNAs (mRNAs) that are produced in individuals with HD compared to those unaffected by the disease. Recall that mRNAs are transcribed from DNA and form the genetic code that is “read” to produce proteins. The changes seen in the expression profiles of individuals affected by HD suggest that the disease impacts transcription at a global level.

From bench to bedside: ^

Because of the strong evidence linking histone dysregulation to the disrupted transcription seen in HD, scientists have hypothesized that addressing the imbalances in histone modifications may improve the symptoms of HD. Research been particularly focused on a diverse group of molecules known as HDAC inhibitors, which prevent the deacetylases from removing acetyl groups.

SAHA:

As mentioned above, CBP gets “trapped” in NIs which disrupts DNA transcription. Steffan, et al. (2001) looked into the possibility of reversing or preventing CBP aggregation in NIs. CBP functions as a HAT but this function is lost when it is “trapped” in the NIs.  This loss of HAT function (or acetyltransferase activity) may be counteracted by inhibiting HDACs (or reducing deacetylase activity).  In other words, blocking the inhibitor of transcription would make up for the low levels of transcription activators. To test this idea, Steffan, et al. used a Drosophila (fruit fly) model of HD, which was genetically modified to express mutant huntingtin. These HD mutant flies show degeneration of nerve cells and decreased survival rates similar to what is observed in people with HD.

To assess the efficacy of reducing deacetylase activity in treating HD, the flies received food containing various HDAC inhibitors, including sodium butyrate and suberoylanilide hydroxamic acid (SAHA). After treatment, the researchers discovered that the flies fed SAHA showed increased histone acetylation compared to untreated flies as well as a slowing in the progression of neurodegeneration.  SAHA also increased survival rates: 70% of untreated flies showed early death compared to 45% of SAHA-treated flies. The results of this study suggest that mutant huntingtin reduces levels of acetylation and transcription by sequestering co-activators (such as CBP and others) and trapping them into aggregates.

Similar studies have been carried out in mouse models of HD, which are better than invertebrate models in assessing how the disease progresses in humans. In 2003, Hockly et al. found that HD mice that were administered SAHA through their drinking water performed better on the Rotarod, a revolving rod that is used to assess motor coordination, than HD mice that were given a placebo.

These effects of SAHA have also been investigated at the molecular level, an important validation step that confirms that the drug is doing what it is supposed to do in the cells of HD mice. Experiments performed by Mielcarek et al. in 2011 showed that SAHA reduced the levels of HDAC4, a class of histone deacetylases, in the cortex and brain stem of HD mice. Recalling the see-saw model of histone acetylation (see above), lower concentrations of HDAC would be expected to result in higher levels of histone acetlyation and thus transcriptional activation—genes being turned “on.” The potentially positive effects SAHA could be seen in the significant reduction of mutant huntingtin aggregation and the partial restoration of the HD mice’s levels of brain-derived neutrophic factor (BDNF), a protein that is important for the survival and growth of certain neurons and is inhibited in HD. (For more information on BDNF, click here.) Despite these promising results, the investigators also found that both wild-type and HD mice treated with SAHA showed significant weight loss, a side-effect that needs to be taken into account if the chemical enters clinical trials.

HDACi 4b:

Thomas et al. (2008): Researchers at The Scripps Research Institute in California have developed an HDAC inhibitor that staves off disease progression in a mouse model of HD. HD mice treated with the drug, called HDACi (HDAC inhibitor) 4b, had significant improvements in movement and coordination, and lost less weight. When scientists looked at the brains of the HD mice, they found that HDACi 4b countered some of the negative effects that HD causes. HD mice usually have a smaller striatum and larger ventricles, and have smaller brains overall. (For more information on HD’s effect on the brain, click here.) However, HD mice treated with HDACi 4b had brains that were just as large as the brains of normal mice, and had a normal-sized striatum and ventricles.

Notably, HDACi 4b has very low toxicity – which is important because many HDAC inhibitors had toxic side effects in mice, and therefore can’t be used in humans. The researchers suggest that HDACi 4b should be studied in humans, as it shows potential to help people with Huntington’s disease.

The results of these studies have been replicated and adapted in subsequent studies looking at other HDAC inhibitors. Taken together, the body of evidence presents a compelling case that this class of molecules could be effective in treating HD. However, there are significant challenges in translating basic science research into clinical therapies. (For more information on clinical trials, click here.)  In 2006, researchers concluded Phase II of a clinical trial examining the safety and tolerability of phenylbutyrate, a HDAC inhibitor that has been found to increase acetylation and improve motor function in mice models of HD. As of the end of 2011, it is unclear whether phenylbutyrate is still actively being considered as a treatment for HD, although scientists have been publishing based on the results of the Phase II study as recently as 2010.

The development of HDAC inhibitors into viable drugs to treat HD can draw from ongoing research into their use as therapies for cancer. Two HDAC inhibitors (including SAHA, the molecule tested in fly and mouse models of HD) are FDA-approved for the treatment of cutaneous T-cell lymphoma, a cancer of a class of immune cells. Many more are being investigated in clinical trials for a range of cancers, an encouraging indication that these drugs are regarded as generally safe for use. But despite their use in current treatments, scientists are still unclear about how increasing histone acetylation improves clinical outcomes. For example, there is now evidence that the majority of molecules targeted by HDACs for acetyl group removal are not histones, but other protein complexes important for cellular function. Clearly, scientists still have an incomplete understanding of how HDAC inhibitors work at the molecular level.

While further research is needed to determine whether HDAC inhibitors can be developed into a safe and effective drug for HD, early results suggest that these molecules hold promise in improving the lives of individuals affected by this terrible disease.

For further reading^

  1. Steffan, et al. “Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila”. 2001. Nature 413: 739-743.
    Treatment with HDAC inhibitors increased survival rates in a Drosophila model of HD.
  2. Thomas EA, Coppola G, Desplats PA, Tang B, Soragni E, Burnett R, Gao F, Fitzgerald KM, Borok JF, Herman D, Geschwind DH, Gottesfeld JM. The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington’s disease transgenic mice. Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15564-9. Epub 2008 Sep 30. A technical article describing the effect of HDACi 4b on HD mice
  3. Gray SG. Targeting Huntington’s disease through histone deacetylases. 2011. Clinical Epigenetics 2: 257-277.
    This technical article provides a broad review of histone deacetylases and their role in Huntington’s disease.
  4. Butler R, Bates G. Histone deacetylase inhibitors as therapeutics for polyglutamine disorders. 2006. Nature Reviews Neuroscience 7:785-796.
    This is another technical review of how histone deacetylase inhibitors may prove useful in treating diseases such as HD.
  5. Kouzarides T. Chromatin Modifications and Their Function. 2007. Cell 128:693-705
    This review article provides a good overview of how the addition and removal of different groups affect chromatin structure and function.
  6. Hockly E, Richon VM, Woodman B et al. 2003. Proceedings of the National Academy of Sciences. 100(4): 2041-2046.
    This is an article from the primary literature about testing SAHA on mice models of HD. that is targeted towards individuals with a science background. Although it may be difficult to understand, the introduction and conclusion are readable.
  7. Mielcarek M, Benn CL, Franklin SA et al. 2011. PLoS ONE 6(11): 1-10.
    This is another article from the primary literature about SAHA in mice models of HD.

    -E. Tan, 11-21-01, updated by M. Hedlin, 10-6-11, Y. Lu, 2-27-12
    -Histone update by Y. Lu, 2-27-12

    From Bench to Bedside