Genetic instructions

Genetic instructions

Introduction:

The genetic instructions used in the development and functioning of all known living organisms are contained within DNA. Within eukaryotes, the genomic DNA is packaged with histone proteins into chromatin, which compacts DNA 10,000-fold (Grant P., 2001). The nucleosome is the fundamental unit of chromatin, composed of an octamer of the four core histones (H3, H4, H2A, H2B) around which 147 base pairs of DNA are wrapped (Kouzarides T., 2007). The core histone molecules possess an N-terminal “tail”, which is unstructured, and is subjected to a large variety of different posttranslational modifications. Chromatin undergoes dynamical changes, jumping between its heterochromatin (condensed) and euchromatin (decondensed) states. Heterochromatin is the tightly packed state of DNA, which limits DNA accessibility and transcription. On the other hand, euchromatin is the lightly packed state of DNA which allows for the active accessibility of genes for transcription. The accessibility of DNA has been characterized as one of the major levels of regulation involved in gene expression, alongside transcription, translation, and posttranslational modifications of proteins. This fourth level of regulation has been dubbed “epigenetics”, which is defined as heritable changes to the transcriptome that are independent of changes in the genome (Spannhoff et al., 2009). The major biochemical modifications that govern epigenetics are DNA methylation and posttranslational histone modifications. To date several distinct histone modifications have been identified: (de)acetylation, (de)methylation, phosphorylation, ubiquitylation, sumoylation, ADP rigosylation, deimination, and proline isomerization (Kouzarides T., 2007). There is strong evidence that these modifications play an important role in the maintenance of transcription as well as in the development of certain diseases, including cancer.

The majority of these modifications are poorly understood, but in recent years there has been a considerable progress in the characterization of acetylation and methylation (Table. 1). Histone acetylation and deacetylation occur on the lysine residues in the N-terminal tail. These reactions are catalyzed by enzymes of the histone acetyltransferase (HAT) or histone deacetylase (HDAC) families. The mode of action of acetylation is neutralization of the positive charge of the histones by adding an acetyl-group to the N-terminal tail, thereby reducing the affinity between histones tails and DNA backbone. This ensures the relaxation of the chromatin structure (euchromatin state) associated with chromatin accessibility and transcriptional activity. Acetyltransferases have been grouped into three main families, GNAT, MYST and CBP/p300 (Sterner and Berger, 2000). These enzymes have been characterized as being able to modify various lysine residues, and limited specificity has been detected for these enzymes. The removal of the acetyl-group from the N-terminal tail (deacetylation) correlates with transcriptional repression. To date, eighteen different human HDACs have been described and these can be divided into four classes based on structural homologies between human and distinct yeast HDAC's (HDAC class I - class IV) (Ellis et al., 2009). These enzymes are involved in numerous signaling pathways and have been observed to be present in a variety of repressive chromatin complexes. Furthermore, the HDAC's do not appear to show much specificity towards particular acetyl groups, although specificity towards particular histones has been observed in their yeast homologues (Hda1 for H3 and H2B; Hos2 for H3 and H4; Kouzarides T., 2007).

Of all the enzymes that modify histones, the histone methyltransferases (HMTs) are one of the most specific. This is perhaps also why methylation is the most characterized modification to date. Unlike with acetylation, where lysine acetylation correlates with expression and deacetylation with repression, lysine methylation can have different effects depending on which residue is modified. In general, methylation at histone H3 lysine 4 (H3K4), H3K36, and H3K79 is associated with transcriptional activation, whereas H3K9, H3K27, and H3K20 methylation correlates with transcriptional repression (Bernstein et al., 2007). Furthermore, HMTs are capable of performing mono-, di-, or tri-methylations at specific lysine residues of histones. The number of methyl-groups being attached to the lysine residues also impacts the transcriptional activity. Mono-methylation of H3K27, H3K9, H4K20, H3K79, and H2BK5 are linked with gene activation, while tri-methylations of H3K27, H3K9, and H3K79 are associated with repression (Ellis et al., 2009). The removal of the methyl-group from the lysine residues is under the control of histone demethylases (HDMTs). To date two types of demethylase domains, with distinct catalytic reactions, have been identified: the LSD1 domain and the JmjC domain. Although the precise function of these demethylases is not fully understood, it is clear that they antagonize methylation by being delivered to the right place at the right time (Yamane et al., 2006). It has been observed that demethylases are selective for mono-, di-, or tri-methylated lysines, which allows for a larger functional control of lysine methylation (Shi and Whetstine, 2007).

Posttranslational modifications to histones, such as (de)methylation and (de)acetylation, are involved in altering gene expression and changes in cellular behavior. The aberrant gene expression and altered patterns of epigenetic modifications are common in human diseases, including cancer (Esteller M., 2007). Cancer can be induced by the aberrant activation of oncogenes and the silencing of tumorsuppressor genes, which can be caused by genetic mutations, as well as by epigenetic modifications. Since epigenetic modifications are reversible, the use of epigenetic drugs in cancer therapy is of major interest in cancer research. Currently available epigenetic drugs posses several drawbacks: 1) their lack of specificity, 2) their transience, and 3) they are only capable to upregulate genes. Using epigenetic editing, it should be possible to develop a treatment that can target specific genes, have a long lasting effect, and the capability to upregulate and/or repress gene expression. Our proposed research will serve as a stepping stone to this ultimate goal. We will focus on the identification of histone effector domains, which can induce specific histone modifications involved in the regulation of gene expression. Furthermore, these effector domains will be fused to specific DNA binding domains to ensure specific targeting of genes.

Methods:

The first stage of this research project will involve the identification of several histone effector domains using the available literature. Although various different histone modifications have been characterized to date, our focus was set on histone acetylation, methylation, and demethylation, since these are enzymes that show great promise in the upregulation of gene expression. The resulting histone effector domains of our first choice are described in Table 1.

Cloning and isolation of the effector domain(s)

Using the list of histone effector domains, at least one effector domain is selected and its cDNA will be cloned into a vector of choice. Selection of an effector domain will depend on the type of histone modification (acetylation, methylation, or demethylation) necessary to induce an upregulation of our gene of interest. The cloned construct will possess the effector domain under influence of a eukaryotic promoter. Furthermore we propose to fuse a specific (His, myc or FLAG) tag to the effector domain, to facilitate protein purification.

Having verified that the DNA sequence of the construct is correct, the construct will be isolated and transfected into a bacterial cell line that will allow for expression of the effector domain. Protein isolation and purification will be achieved by affinity chromatography.

In vitro analysis of effector domain(s) functionality

To determine the activity of the truncated purified effector domain there are several of in vitro assays that can be used. For a histone acetyltransferase (HAT) effect domain there are several commercially available colorimetrical kits, such as the Active Motif's fluorescent HAT Assay Kit, that can determine whether or not acetylation was induced by the purified protein (truncated effector domain). Histone methyltransferases (HMTs) or histone demethylases (HDMTs) activity can be measured via isotopically labeled methyl-3H and/or identifying modified histones by purified effector domains using fluography (as described in Yuan et al., 2008 and Trojer et al., 2008). To determine if the desired histone modification has been induced by the purified protein the commercially available Active Motif's Chromatin Assembly Kit could be used followed by an in vitro chromatin immunoprecipitation (ChIP). The Active Motif's Chromatin Assembly Kit allows for the assembly of an artificial chromatin structure composed of a DNA sequence of interest, cloned into a vector, together with purified histones of interest (as described by Kundu et al., 2000). The assembled chromatin structure is incubated with the purified effector domain, and the histone modifications can be identified via a ChIP using modification specific antibodies.

Table 1. Histone-Modifying Enzymes

Enzymes that modify Histones

Effector Domain

Residues

Modification type

Activation/repression

Methyltransferases

SetDB1

SET

H3K9

Mono-methylation

Activation

ATXR5

SET

H3K27

Mono-methylation

Activation

Set7/9

SET

H3K4

Mono-methylation

Activation

Set1

SET

H3K4

Di-methylation

Activation

MLL1

SET

H3K4

Tri-methylation

Activation

PR-Set7/KMT5A

SET

H4K20

Mono-methylation

Activation

NSD2

SET

H3K36

Tri-methylation

activation

Dot1

SET

H3K79

Mono-methylation

activation

Dot1

SET

H3K79

Tri-methylation

Repression, activation

Demethylases

LSD1

LSD1

H3K4

Mono-, Di-methylation

Repression

JHDM1A

JmjC

H3K36

Mono-, Di-methylation

Activation

JMJD1A

JmjC

H3K9

Mono-, Di-methylation

Repression, activation

JMJD2A

JmjC

H3K9

Tri-, Di-methylation

Activation

H3K36

Tri-, Di-methylation

Repression, activation

JMJD2B

JmjC

H3K9

Tri-, Di-methylation

Activation

JMJD2C

JmjC

H3K9

Tri-, Di-methylation

Activation

H3K36

Tri-, Di-methylation

Repression, activation

JMJD2D

JmjC

H3K9

Tri-, Di-, Mono-methylation

Activation, activation, repression

Acetyltransferase

HAT1

HAT

H4K5, H4K12

Acetylation

Activation

CBP/P300

HAT

H3K14, H3K18, H4K5, H4K8, H2AK5, H2BK12, H2BK15

Acetylation

Activation

PCAF/GCN5

HAT

H3K9, H3K14, H3K18

Acetylation

Activation

TIP60

HAT

H4K5, H4K8, H4K12, H4K16, H3K14

Acetylation

Activation

HB01

HAT

H4K5, H4K8, H4K12, H4K16, H3K14

Acetylation

Activation

ScSAS3

HAT

H3K14, H3K23

Acetylation

Activation

ScSAS2

HAT

H4K16

Acetylation

Activation

ScRTT109

HAT

H3K56

Acetylation

Activation

Fusing effector domain to specific zinc finger complex

Following the in vitro analysis of the effector domain and its capability to induce histone modifications, a fusion between the effector domain and a zinc-finger complex will be constructed. The zinc-finger complex will be used as a DNA binding domain specifically targeted to the gene of interest. Six zinc-finger modules will make up the zinc-finger complex, allowing for a unique DNA binding motif (Dr. Marianne G. Rots, personal communication). The sequence for the fusion protein will be cloned into a vector of choice.

Ex vivo analysis of fusion protein

The cloned construct will be isolated and transfected into a cell line of choice, preferentially a cancer cell line containing the down regulated state of our tumor suppressor gene of interest. Transfection will be followed by a ChIP assay using modification specific antibodies.

If the presence of the specific histone modifications is observed in the transfected cells, then the gene expression will be analyzed with a western blot. This will allow us to identify the presences of both the fusion protein and of the tumor suppressor protein of interest. To quantify the expression of the gene of interest, which is induced by the fusion protein, a qRT-PCR will be performed.

Finally, if the transfected cell lines have shown to express the tumorsuppressor gene of interest, a phenotypical analysis will be performed, comparing them to the wild-type cell line.

Milestones and deliverables:

The proposed research project is set to use novel techniques in identifying the mode of action of various effector domains from histone modification enzymes, and their realistic effectiveness as tumorsuppressor gene regulators. A realistic time schedule has been depicted in Figure 1, including all the steps involved in achieving the projects goal. There are certain milestones that have to be achieved for the success of this project:

  1. Characterization of in vitro activity of a truncated epigenetic effector domain.
  2. Characterization of in vitro activity of the epigenetic effector domain and zinc-finger complex fusion protein.
  3. Ex vivo characterization of gene specific activation by the fusion protein.

Although the various stages of the research project are straight forward, there are some stages where some complications might be encountered. One major complication might arise while creating the fusion construct of the truncated effector domain with the zinc-finger complex. Fusing these two on a genetic level may be attainable, but problems may arise in attaining the correct folding of the eventual fusion protein. Furthermore, identifying which effector domain possesses the best enzymatic activity, in vitro and/or ex vivo, necessary for a specific histone modification may be quit time consuming.

Importance of the proposed research

Changes to the epigenetic code have been identified as having a causative effect on numerous diseases, including several types of cancer. More importantly these epigenetic modifications are reversible, and thus identifying enzymes capable of inducing specific epigenetic modifications is of great importance to treating these diseases. Not only is the identification of epigenetic modification enzymes necessary, but being able to target these enzymes to induce specific modifications to regulate specific gene expression is of tremendous importance. Our proposed research will demonstrate that it is possible to target specific sites for histone modifications using fusion proteins comprised of a histone modification effector domain and a zinc-finger complex (made up of six zinc-finger modules). Such a proposed fusion protein will be able to effectively and permanently regulate the expression of specific disease-linked genes. Such a revolutionary treatment would have great impact on the lives of the patients being treated. Unlike conventional drug treatment therapies, epigenetic treatments should result in a drastic decrease in side effects, since only the causative gene(s) will be targeted.

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