Contact: Paul Preuss, [email protected]

Intricately wound, folded, and looped chromatin (blue) meets chromatin-remodeling and modifying factors at sites on a cage-like structure formed by SATB1 proteins (gold). In this image the chromatin is densely packed heterochromatin, a type associated with silent genes. (Image: Abby Dernburg)

A mammalian body contains trillions of cells, most of them packed with a whole genome’s worth of DNA. Stretched out straight, the DNA in the nucleus of just one cell would be a yard or two long. How does it all fit?

Through tight, intricate, twisting and folding: a thread of DNA winds around a spool made of proteins called histones; thread and spool together make a nucleosome. The DNA strings the nucleosomes together like beads, and the beads clump together in thick fibers; the fibers fold into loops, and the loops are further looped into the ropy mass of chromatin of which the individual chromosomes in the nucleus are made.

So many levels of winding, folding, and looping create a dilemma: for a cell to express proteins, it needs to transcribe genes, which requires double-stranded DNA to unzip where the gene is encoded. DNA wound up tight in chromatin can’t unzip; like the wire in a coiled steel cable, most of it can’t even be reached.

Researchers led by Terumi Kohwi-Shigematsu of Berkeley Lab’s Life Sciences Division are learning the secrets of how specific sites of DNA in the genome can be made accessible for protein factors that change the chromatin structure locally. These changes make gene transcription possible or repress it; in this way, at appropriate times and places, specific sets of genes are expressed or remain silent, and each type of cell expresses only the genes appropriate to its physiological role.

Investigating unusual DNA structures

A decade ago Kohwi-Shigematsu and her husband, Yoshinori Kohwi, also in Berkeley Lab’s Life Sciences Division, were investigating certain DNA sequences with a strong tendency to adopt noncanonical structures — ones inclined to coil not quite “by the book.”

They identified a special class of sequences with a strong tendency to pop open — and also to unzip the neighboring sequences, when the DNA helix is under negative supercoiling — that is, when the intact double strand of DNA is coiled in the opposite direction from the way the two strands coil around each other. They called these sequences “base unpairing regions,” or BURs.

BURs under negative supercoiling tend to close up and become double stranded if the microenvironment gets saltier. But short core sequences, a few bases long, refuse to pair up no matter how salty the surroundings.

BURs are rich in the bases adenine and thymine (A and T), which pair only with each other (as do the other two DNA bases, cytosine and guanine, C and G). While sequences rich in A and T separate a bit more easily into single strands than C- and G-rich sequences, not just any stretch of As and Ts readily unzips.

Base unpairing regions, however, contain clusters of ATC sequences where only well-mixed As, Ts, and Cs occur on one strand. Kohwi and Kohwi-Shigematsu called such a cluster an ATC sequence context.

“We reasoned that if these regions were biologically important, there must be an important protein associated with them,” says Kohwi-Shigematsu. Using cloned BURs as bait, they went fishing in a library of proteins and hooked a big one, which they straightforwardly named “special AT-rich binding protein 1,” better known as SATB1.

Although SATB1 is very particular about latching onto base unpairing regions, it does not attach itself to exposed DNA bases; instead, it slides into the minor groove on the outside of double-stranded BUR sequences. Rather than recognizing a particular primary sequence, SATB1 recognizes the ATC sequence context, a likely site for base unpairing. Thus SATB1 manages to be both specific and versatile at the same time.

In a strong salt solution the cell nucleus bursts and chromatin spills out. But even in very strong solutions not all proteins are removed.

BURs are often found in matrix attachment regions, operationally defined as genomic DNA sequences tethered to the nuclear components that resist salt extraction.

Arming the immune system

Matrix attachment regions in general bind to several proteins, most found in many different cell types. SATB1 works only in a few distinct kinds of cells (including the embryonic stem cells much in the news), all of which are unspecialized precursors of mature cells that later assume particular functions. SATB1 is most widespread in the cells known as thymocytes.

Thymocytes, so named because they grow to maturity in the thymus gland, are the precursors of T cells, among the immune system’s most potent weapons. “Killer” T cells (cytotoxic lymphocytes) go straight for the metaphorical jugular of invading disease organisms, tumors, or other cells marked for destruction. “Helper” T cells emit proteins like interleukin 2 that help identify targets, stimulate the defenders, and aid in the attack. (Helper T cells are themselves a principal target of HIV infection.)

Mature killer and helper T cells are distinguished by cell-surface markers designated CD8 and CD4. Early in their development, thymocytes have neither of these markers. They proliferate rapidly and differentiate into a double-positive stage, expressing both CD4 and CD8.

During the double-positive stage, cells that are useless or “self-reactive” — having an unfortunate tendency to kill the host — are eliminated in droves; approximately 98 percent of the thymocytes generated each day die without leaving the thymus. Survivors become “single positive” for either CD4, as mature helper T cells, or CD8, as mature killer T cells.

Kohwi-Shigematsu and her colleagues soon learned that SATB1 plays a crucial role in T-cell development.

Down for the count

Unlike this “wild-type” lab mouse, knockout mice lack specific genes, and thus cannot express the proteins for which they code.

Kohwi-Shigematsu and her colleagues pinpointed SATB1’s vital role in T-cell development by preparing “knockout” mice that were genetically normal in all respects except that they lacked the gene for SATB1, and thus the SATB1 protein itself.

Designated SATB1-null, the knockout mice had problems from the start. Some of their reflexive behaviors were odd. Although most of their organs were normal, their thymus, spleen, and lymph nodes were smaller than those of the normal “wild” type. They were small and thin and lived only three weeks; wild-type mice live two years or more.

Physiological causes soon emerged: lack of SATB1 had wrecked the immune system of the knockout mice. Very few immature thymocytes were produced to begin with, and those that progressed to the crucial double-positive stage inappropriately expressed receptors and markers. It was at this stage that most T-cell development in the SATB1-null mice came to a halt; while a very low number of helper T cells were produced, killer T cells were absent.

Thymocytes that did develop to the double-positive stage expressed proteins, like interleukin-2 receptors, that should normally be repressed at this stage. Worse, the signals that activate normal mature T cells, inducing them to produce interleukin-2 and other proteins, instead caused the few helper T cells from SATB1 knockout mice to die off through apoptosis, programmed cell suicide.

In wild-type mice (columns marked WT), expression of certain interleukin receptor proteins is repressed during T-cell development. But in knockout mice with no SATB1 (columns marked KO), these proteins are expressed ectopically — literally “out of place.”

Numerous genes essential to T-cell development and function were unregulated or misregulated, including genes for interleukin receptors, a gene for a chemokine receptors that stimulates activity in immune-system cells, genes that code for DNA binding proteins, genes for cell-surface markers, and genes related to apoptosis and cell proliferation. Nor is expression of SATB1 restricted to thymocytes: it is also found in other specific precursor/progenitor cells, including osteoblast cells that become bone. Runaway apoptosis of cells that normally express SATB1 is what may have killed the knockout mice at the end of their short lives.

“In the absence of SATB1, hundreds of genes are dysregulated in thymocytes,” says Kohwi-Shigematsu. “SATB1 tells the cells when and which genes should be expressed or remain silent.” In thymocytes, SATB1 appears to regulate up to two percent of all the genes in the mouse genome.

An architect of the chromatin

Evidence for what began as a hypothesis by Kohwi-Shigematsu and her colleagues was fast accumulating: SATB1 forms a physical network inside the nucleus of thymocytes, one that resists salt extraction and the removal of genomic DNA and RNA.

The SATB1 network actively tethers specialized DNA sequences and organizes the chromosomes into distinct loop domains, containing specific sets of genes and other DNA. SATB1 is not just a chromosome organizer, however. It orchestrates gene expression by allowing chromatin remodeling to take place at specific genomic locations.

To find the proteins that associated with SATB1 at specific gene locations, Kohwi-Shigematsu and her colleagues applied techniques with arcane names like DNA-affinity chromatography and chromatin immunoprecipitation. What they found were protein complexes known to be involved in chromatin remodeling and gene regulation.

These included ACF1, “ATP-utilizing chromatin assembly and remodeling factor 1,” and ISWI, “imitation switch,” named for its resemblance to yeast’s SWI switch, a component of one of the first-identified molecular machines for remodeling chromatin. ACF1 and ISWI are members of CHRAC, the “chromosome accessibility complex,” which shuttles nucleosome beads back and forth like an accountant manipulating an abacus.

Another complex, the “nucleosome remodeling and histone deacetylation” (NuRD) complex, works directly on the histone units at the cores of nucleosome beads. Histone proteins, rather like New World monkeys, have prehensile “tails” with which they bind themselves together, eight to a nucleosome spool, or bind the DNA to the spool, or bind the nucleosome to other nucleosomes and different kinds of histones. Histone tails grasp tightly if deacetylated (lacking a particular group of carbon, hydrogen, and oxygen atoms); when acetylated, they let go — a necessary step in allowing DNA to be read by enzymes for gene transcription.

The “tails” of deacetylated histone proteins bind DNA in chromatin (left). When acetylated, the tails let go — a necessary step in allowing DNA strands to be read for gene transcription.

The researchers have shown, explicitly in the case of the interleukin-2 receptor-alpha gene (IL-2Ra) and in other genes as well, that SATB1 controls gene expression — in some cases up to tens of thousands of bases away from the attachment site — by providing docking sites for these remodeling complexes, which alter the shape of nucleosome arrays and activate and deactivate DNA transcription.

In microscope images collected with the aid of Life Sciences researcher Abby Dernburg, the network in the cell nucleus stands out clearly as a cage-like structure of fluorescently stained SATB1 proteins circumscribing heterochromatin. (Heterochromatin is a densely packed type of chromatin associated with silent genes.) The cage-like SATB1 structure provides the sites where the bases of the looped chromatin meet chromatin-remodeling and modifying factors, for which it functions as a platform.

How does SATB1 know whether to activate or repress transcription of a gene at a given time? Whether chromatin remodeling is motivated by demand from below or orders from above, Kohwi-Shigematsu says, “is a chicken and egg question” — one that will keep her and her colleagues busy for the foreseeable future, in the search for even deeper secrets of gene regulation.

Additional information