March 15, 2015 (Vol. 35, No. 6)

Richard A. A. Stein M.D., Ph.D.

Researchers Dare to Break Open the Black Box of Gene Expression.

Over 70 years ago, quantum physicist Erwin Schrödinger offered his thoughts on biology’s physical underpinnings, inspiring a generation of molecular biologists by suggesting that genes were information carriers. He even referred to chromosomes as being the “architect’s plan and builder’s craft in one.”

Posterity has shown that Schrödinger was clearer about the plan than the craft. The plan, he said, would likely depend on an “aperiodic crystal” or a kind of “code-script.” This code-script has come to be identified with DNA and its variable base sequence. Schrödinger, however, did not explain how code-script might be copied or how genes might be expressed.

The genetic thread has since been picked up by scientists intent on elucidating chromosomal craft. These scientists have focused on deciphering the organization and the regulation of genetic material. They are, at present, redoubling their efforts, using the tools of biotechnology and erecting interdisciplinary frameworks.

“Two areas that have long been important, and continue to be so, are the robust identification of biologically important targets of protein and RNA regulators of gene expression and the integration of single-gene and whole-genome studies with systems biology network analyses,” says H. Robert Horvitz, Ph.D., professor of biology at MIT and co-recipient of the 2002 Nobel Prize in Physiology or Medicine.

Apoptosis Pathways

A major research effort in Dr. Horvitz’s laboratory is focusing on apoptosis or programmed cell death, a process that plays fundamental roles during development, tissue homeostasis, and disease, to ensure that unnecessary or pathologically modified cells are eliminated. The laboratory has even uncovered possibilities for alternative apoptosis pathways.

Dr. Horvitz and colleagues arrived at key insights after investigating GCN-1 and ABCF-3, two translational regulators from the roundworm Caenorhabditis elegans. The investigators found that the two proteins, which are involved in translational initiation, form a complex in vivo, and are evolutionarily conserved, orchestrate in most somatic cells a pathway that is distinct from the canonical apoptotic pathway.

The prevailing view has been that apoptosis is regulated by CED-9, the mammalian BCL-2 homolog. Apoptosis regulated by GCN-1 and ABCF-3, however, opens the possibility of capitalizing on newly discovered similarities and differences across species. Scientists may dissect new cellular and molecular pathways of cell death, and they may even find that a higher level regulatory logic is conserved among species.

“Studies that draw upon rather than ignore findings from multiple organisms can progress most rapidly and effectively,” asserts Dr. Horvitz. “The elucidation of the RTK-Ras-MAP kinase pathway, which [relied on] studies of the development of the sexual organ of a worm and the eye of a fly in combination with studies of signal transduction by mammalian cells in culture, provides a striking example.”

As part of apoptotic pathways, the involvement of caspases in many cellular processes, including cell death, inflammation, and lymphocyte maturation, helped explain the molecular events involved and opened novel therapeutic avenues. “Two areas that intrigue me are identifying caspase cleavage products that are the real drivers of apoptotic death and understanding the mechanisms of caspase-independent apoptotic death,” comments Dr. Horvitz. While caspase-dependent programmed cell death has attracted more attention, caspase-independent pathways are emerging as a novel research area.

Dr. Horvitz and colleagues reported that in C. elegans mutants lacking all four caspase genes, embryonic cells with apoptosis-like morphological characteristics, are still extruded into the extra-embryonic space. This finding revealed that the roundworm is still able to orchestrate apoptosis, even in the absence of caspases. Several proteins, including the C. elegans homologs of the human tumor suppressor kinase LKB1, were required for this newly unveiled caspase-independent cell shedding.

In humans, LKB1 is mutated in Peutz-Jeghers syndrome, a disease characterized by noncancerous gastrointestinal polyps that develop early in life, and an increased risk of malignant changes in multiple tissues over time. This suggests that polyp formation in some patients with Peutz-Jeghers syndrome might result from defects in a process of apoptotic epithelial cell extrusion mechanistically like that seen in C. elegans, a finding that could clarify the pathological processes and assist in developing prophylactic and therapeutic interventions.

Nucleosome Dynamics

“For the first time, we were able to look at histones genome-wide at the subnucleosomal level,” says B. Franklin Pugh, Ph.D., professor of biochemistry and molecular biology and chair of the department of molecular biology at Penn State University. The organization of the eukaryotic genetic material into nucleosomes, in which DNA is wrapped around histone octamer cores, has intrigued scientists for decades. Scientists have been especially interested in nuclesome dynamics and the mechanistic implications for DNA replication, transcription, and recombination.

“Nucleosomes are not simply beads on the string,” emphasizes Dr. Pugh. “They are dynamic and come apart in many different ways.”

Even though nucleosomes have a two-fold symmetry of histone organization, the manner in which RNA polymerase engages the nucleosome-free promoter region of transcribed genes, a key process during transcription, is asymmetrical. Moreover, as a result of histone epigenetic marks such as methylation, acetylation, ubiquitination, and phosphorylation, chemical activities may be asymmetric or distinct on different sides of the nucleosome.

Using a chromatin immunoprecipitation-exonuclease (ChIP-exo) assay, Dr. Pugh and colleagues profiled nucleosome structure and symmetry at approximately 60,000 sites around the budding yeast genome. This work revealed that nucleosomes are asymmetric with respect to gene transcription, and helped define and characterize several asymmetric features on the first nucleosome that encounters RNA polymerase during transcription.

“Knowing that this asymmetry exists within nucleosomes is important, but we still don’t know how it impacts transcription,” admits Dr. Pugh. The data also points toward the in vivo existence of half-nucleosomes, which present only one copy of each histone. This finding is consistent with earlier observations from biochemical studies.

Research conducted in the past two decades has not only revealed more about the structural and functional roles histones play in organizing eukaryotic genetic material, it has also unveiled many additional proteins that regulate gene expression. Historically, many studies relied on biochemical and genetic studies that used single genes as experimental models.

“We still don’t know how all these proteins work together, and figuring it out will be challenging,” concludes Dr. Pugh. “The next step is to understand, at the genomic level, how individual genes are regulated and characterize the common themes of this regulation.”

Chromatin Remodelers

One of the major challenges in understanding the dynamic organization of the genetic material inside cells involves explaining how 3 billion base pairs of DNA can be compactly organized into domains, yet become accessible for processes including transcription, translation, and recombination. The balance between various states of the genome is accomplished through chromatin transitions, which are mediated by the dynamic action of specialized protein complexes.

“Chromatin remodeling proteins do many different things, and the net result is that at particular regulatory sites or promoters they reorganize the DNA template in ways that we don’t really understand yet,” says Gordon L. Hager, Ph.D., chief at the Laboratory of Receptor Biology and Gene Expression of the National Institutes of Health/National Cancer Institute.

Several approaches are currently used to capture the interaction between DNA and chromatin regulators. Genomics studies present the disadvantage that the information is collected from large populations of nonviable cells.

“Because every cell is in a different state, those populations are heterogeneous, and the results provide snapshots of a wide diversity of states across the population,” explains Dr. Hager. Live cells provide a more faithful representation of real-time processes, but the low resolution in visualizing individual genomic sites is a major limitation.

Studies on chromatin remodeling present the advantage that they capture biochemical processes. “But it is still complicated to incorporate every process,” adds Dr. Hager. “All these approaches have their weak points.”

To map the interactions between chromatin remodeling complexes and the DNA on a genome-wide scale in mouse mammary epithelial cells, Dr. Hager and colleagues used ChIP-Seq to interrogate the distribution of three different remodelers. The proteins that were profiled included the Brg1 subunit of the SWI/SNF complex, Snf2h, and Chd4, which were reported in the literature to perform unique roles in regulating chromatin structure.

“The first thing we discovered was that these remodelers are widely distributed in the genome,” recalls Dr. Hager. About 60% of the binding sites for these proteins were in the gene promoters and bodies, and about 40% occupied intergenic regions.

Pairwise comparisons that examined genomic sites occupied by each of these remodelers revealed that any given genomic site may have more than one remodeler, and that some of them may have all three. Furthermore, some sites may be co-occupied by remodeling complexes with antagonistic actions, such as a complex that can open and a complex that can close the chromatin.

To better understand the functional interaction of remodelers with specific genomic spots, Dr. Hager and colleagues generated dominant negative knockout cell lines. In these lines, ATP-dependent remodelers were individually inactivated by mutations in conserved residues of their ATPase domains. “This allowed us to ask, at any given site, not only whether the protein is present but also whether it is doing something,” says Dr. Hager.

This strategy showed that when multiple remodeler protein complexes are present at any single site, they are each functional. Overall, the findings point toward a model in which transcription factors and remodelers cycle through a complex series of states.

Although studying the dynamic interaction between regulatory complexes and DNA has been technologically challenging, the newly developed single-molecule tracking approach, in which proteins are labeled with tags and visualized with fluorescent chemicals, allows live processes to be seen with higher resolution. “This approach is resistant to photobleaching, and allows single molecules to be visualized as they move around the cell,” notes Dr. Hager. Single-molecule tracking experiments revealed that there are two types of stops as remodeling complexes move around DNA templates.

“Very fast stops, which last about half a second, are nonspecific events, and slow stops, which last five to ten seconds, represent real binding and regulatory events,” informs Dr. Hager. These results support and extend findings that originated several years ago from Dr. Hager’s lab. A landmark study that utilized recovery from photobleaching revealed that the glucocorticoid receptor was bound to its regulatory sites for only approximately 10 seconds.

“Many transcription factors are moving on and off the template very rapidly, despite their very high affinity for their binding sites in vitro,” concludes Dr. Hager. “Thus, a central question in current research becomes how these sites are regulated and how accessibility is being controlled.”

Researchers at the National Cancer Institute have interrogated the distribution of remodeler complexes, large ATP-dependent enzymes that rearrange the local nucleosome structure to modulate access of site-specific DNA binding proteins to the template. These complexes are directed to specific sites in the genome both by transcription factor interactions and by interactions with modified histones. Multiple complexes can operate at a given site, either working together to increase and modify accessibility (synergy) or functioning against each other in an antagonistic mode.

Monallelic Gene Expression

“We became interested in evaluating whether random autosomal monoallelic gene expression, originally studied in gene families expressed in the nervous system, might be a more general theme that is carried out throughout the genome in other cell types,” says David L. Spector, Ph.D., professor and director of research at the Cold Spring Harbor Laboratory. Random autosomal monoallelic gene expression refers to the phenomenon in which transcription of a gene occurs from only one of its two homologous alleles, the second one being silent.

Historically, X-chromosome inactivation that occurs during the blastocyst stage in most female mammals is the most widely known example of monoallelic gene expression. The phenomenon was subsequently described in autosomal cells, including genes encoding for the olfactory and immunoglobulin receptors.

Using an allele-specific RNA-sequencing screen, Dr. Spector and colleagues reported that during mouse embryonic stem cell differentiation into neural progenitor cells, an approximately sixfold increase in random autosomal monoallelically expressed genes was observed in the genome. For these genes, specific histone post-translational modifications were differentially enriched between the two alleles.

“We would like to understand whether the massive change in gene expression and in chromatin compaction during differentiation results in monoallelic expression,” states Dr. Spector. During chromatin compaction changes, certain alleles may be positioned in different local neighborhoods in the three-dimensional space of the nucleus.  “An allele that might end up in a different neighborhood could result in its activation or its silencing.”

A somewhat unexpected finding that emerged during this work was that a subset of monoallelically expressed genes may exhibit transcriptional compensation by the active allele, raising the possibility of allele–allele communication. This process is thought to help maintain the global output of the respective genes.

“Being able to study single alleles in single cells is the direction we would like to go both from a genomic and from a cell biological perspective,” comments Dr. Spector. “There are challenges in both of these directions.” 

Expression-Based Cell Sorting

Tissue-capable cell populations are essential for many research and clinical applications, but generating them can be challenging. A particular difficulty is the isolation of cells with specific characteristics from heterogeneous populations. To facilitate the generation of tissue-capable cell populations, researchers at Brown University have introduced a novel approach to cell sorting.

“We developed a method for the gene expression-based enrichment of live cells,” says Eric M. Darling, Ph.D., assistant professor of molecular pharmacology, physiology, and biotechnology and a member of the Center for Biomedical Engineering at Brown University. Dr. Darling and colleagues propose a method that is based on mRNA expression and takes advantage of a molecular beacon, a hairpin-shaped oligodeoxynucleotide that has a fluorophore on the 5′ end and a quencher at the 3′ end.

“This probe is quenched when it is free, but it becomes unquenched when its loop binds the mRNA target,” explains Dr. Darling. Using cells derived from the stromal vascular fraction of adipose tissue, Dr. Darling and colleagues used a molecular beacon for alkaline phosphatase to identify and isolate cellular subpopulations with an enhanced osteogenic potential.

“Cells that are able to become osteoblastic start expressing osteogenic genes,” asserts Dr. Darling. “We can tag them with this marker and separate them out into positive, negative, and unsorted groups.” The peak expression period of osteogenic genes was achieved four days after priming.

While this approach shows promise for research and therapeutic applications, there are several challenges that still need to be overcome. “The difficulty with molecular beacons is that they can have high false-positive rates. Also, there is a lot of noise in the system,” admits Dr. Darling. “But careful design and new materials can help with some of these limitations.”

Gene expression-based sorting conducted at Brown University identified a population of mesenchymal stem cells capable of differentiating along the targeted, osteogenic lineage. ALPL+ cells deposited significantly more calcified matrix than ALPL- or unsorted cells, visualized by Alizarin red staining. In addition to more robust osteogenesis, the sorting process yielded greater cell numbers than surface marker-based approaches.

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