Zheng Lian

A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster

We have identified an intergenic transcriptional activity that is located between the human HOXA1 and HOXA2 genes, shows myeloid-specific expression, and is up-regulated during granulocytic differentiation. The novel gene, termed HOTAIRM1 (HOX antisense intergenic RNA myeloid 1), is transcribed antisense to the HOXA genes and originates from the same CpG island that embeds the start site of HOXA1. The transcript appears to be a noncoding RNA containing no long open-reading frame; sucrose gradient analysis shows no association with polyribosomal fractions. HOTAIRM1 is the most prominent intergenic transcript expressed and up-regulated during induced granulocytic differentiation of NB4 promyelocytic leukemia and normal human hematopoietic cells; its expression is specific to the myeloid lineage. Its induction during retinoic acid (RA)-driven granulocytic differentiation is through RA receptor and may depend on the expression of myeloid cell development factors targeted by RA signaling. Knockdown of HOTAIRM1 quantitatively blunted RA-induced expression of HOXA1 and HOXA4 during the myeloid differentiation of NB4 cells, and selectively attenuated induction of transcripts for the myeloid differentiation genes CD11b and CD18, but did not noticeably impact the more distal HOXA genes. These findings suggest that HOTAIRM1 plays a role in the myelopoiesis through modulation of gene expression in the HOXA cluster.

Gene expression in human neutrophils during activation and priming by bacterial lipopolysaccharide

Circulating neutrophils play a key role both in the systemic inflammatory response and in complications of bacterial infection such as septic shock and septic multiple organ dysfunction syndrome. We have analyzed gene expression patterns in human neutrophils stimulated by E. coli lipopolysaccharide (LPS), with or without prior exposure to LPS, using differential display and oligonucleotide chip techniques. We identified 307 genes that were activated or repressed after treatment with LPS at 10 ng/ml and 385 genes after LPS at 100 ng/ml, compared with untreated neutrophils. The two sets included many transcription factors, cytokines, chemokines, interleukins, and surface antigens, as well as members of the toll‐like receptor, Rel/NF‐κB, and immune mediator gene families. Time course analysis showed that the early and late neutrophil responses to LPS share some common mechanisms, but many changes in gene expression are transient or late to develop. Neutrophils also showed a priming response to LPS, in which 97 genes significantly changed expression on re‐exposure to lower dose LPS and were analyzed by unsupervised hierarchical clustering. These findings indicate that the neutrophil is a transcriptionally active cell responsive to environmental stimuli and capable of a complex series of both early and late changes in gene expression. Supplementary material for this article can be found on the Journal of Cellular Biochemistry website (http://jws‐edci.interscience.wiley.com:8998/jpages/0730‐2312/suppmat/89/v89.page.html). J. Cell. Biochem. 89: 848–861, 2003.

Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells

Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.